Apolipoprotein B-specific monoclonal antibodies produced by two novel hybridomas

ABSTRACT

Two hybridomas that produce receptors containing antibody combining sites that immunoreact with apolipoprotein B-100 are disclosed as are uses for the receptors, compositions and diagnostic systems that include the receptors.

This invention was made with government support under NationalInstitutes of Health Contract HL 14197. The government has certainrights in the invention.

This is a continuation of copending application Ser. No. 06/893,659filed on Aug. 6, 1989, now abandoned.

DESCRIPTION

1. Technical Field of the Invention

The present invention relates generally to novel hybridomas, and morespecifically to hybridomas that produce receptor molecules thatimmunoreact with apolipoprotein B-100, to the receptor molecules soproduced as well as to diagnostic methods and systems employing thereceptor molecules.

2. Background of the Invention

Lipoproteins are the primary carriers of plasma cholesterol andtriglycerides. They are micellar lipid-protein complexes that containprotein (referred to as apoprotein) and polar lipids organized in asurface film that surrounds a neutral lipid (triglyceride andcholesteryl ester) core. Lipoproteins were originally identified basedon their bouyant densities as measured by ultracentrifugation.Accordingly there are four major density classes: chylomicrons, very lowdensity lipoproteins (VLDL), low-density lipoproteins (LDL), andhigh-density lipoproteins (HDL).

Paralleling advances in the technology of ultracentrifugal separationsthere has been a further subdivision of the LDL and HDL density classesinto further subclasses of greater homogeneity. For instance, LDL can beresolved into an intermediate density lipoprotein (IDL) and an LDL₂subclass. However, even these subclasses are composed of functionallyheterogeneous populations of lipoprotein particles because of theirvaried apoprotein content.

Eight major apoproteins, A-I, A-II, A-IV, B, C-I, C-II, C-III, and E,have been isolated, and of the group of minor apoproteins that can berecovered in larger amounts from certain density classes, most can alsobe found in other density classes. Thus, most LDL particles contain onlyapo B, however a few particles also contain other apoproteins and thisaccounts for the trace amounts of apo C-I, apo C-II, C-III, and apo Epresent in this density class.

In some cases, specific functions have been assigned to particularapoproteins. For instance, a species of apo B synthesized in the liver,termed apo B-100, is recognized and bound by cellular LDL receptors. Bybinding apo B-100, these receptors bind LDL particles and extract themfrom the plasma. The LDL is thereby taken into the cells and brokendown, yielding its cholesterol to serve each cell's needs. The apo B-LDLreceptor interaction thus plays a major role in removal of LDLcholesterol from the bloodstream.

Another species of apo B, termed apo B-48, is not recognized by the LDLreceptor. This apo B species, which is only 48 percent as large as apoB-100, is synthesized in humans only by the intestine. Lipoproteinscontaining apo B-48, such as chylomicrons and chylomicron remnants, donot bind to the LDL receptor.

Although these two species of apo B appear to be under separate geneticcontrols (a single patient has been described whose body makes apo B-48,but not apo B-100), immunologic studies have demonstrated thatapoproteins B-100 and B-48 share antigenic determinants. At least threeresearch groups have reported generation of a total of seven differentmonoclonal antibodies that bind to either of apo B-100 and apo B--48.The data reported by those researchers strongly suggest that apo B-48and apo B-100 are structurally related proteins; i.e., that apo B-48 mayrepresent a portion of the apo B-100 protein. Evidence also has beenreported that apo B-48 and apo B-100 are not found on the samelipoprotein particle, suggesting that separate apo B particles exist.

Recently, several investigators have suggested that plasma levels of apoB may be more predictive of coronary artery disease (CAD) risk thanplasma LDL cholesterol levels. Sniderman et al., Proc. Natl. Acad. Sci.USA 77, 604-608 (1980). Because artherosclerotic vascular disease andits complications continue to be the leading cause of death anddebilitation in Western society, there has been a long felt need withinthe biomedical industry for assay systems capable of identifyingindividuals at risk for CAD.

Many types of immunoassays for plasma apoprotein B utilizing specificantibody-containing antisera have been reported, including competitivefluid phase and solid phase radioimmunoassays (RIA), enzyme-linkedimmunosorbant assays (ELISA), radial immunodiffusion assays and others.Problems limiting the widespread application of these apo B immunoassayshave been reproducibility, and the quality and specificity of theantisera used. Reviews of the methodological problems of each of thevarious types of apo B assays are found in Currey et al., Clin. Chem.24, 280-286 (1978) and Rosseneu et al., Clin. Chem. 28, 427-433 (1983).

Several investigators have reported development of panels of monoclonalantibodies against human apo B for use in studying its antigenicstructure and role in lipoprotein metabolism. Furthermore, there havebeen reports of using anti-apo B monoclonal antibodies to measure plasmaapo B levels in fluid-phase RIA's. Patton et al., Clin. Chem. 29,1898-1903 (1983) and Maynard et al., Clin. Chem. 30, 1620-1624 (1984).In addition, one group has reported use of a mixture of anti-apo Bmonoclonal antibodies in a radial immunodiffusion assay for plasma apoB. Marconvina et al., Clin. Chim. Acta 147, 117-125 (1985). However,these assay techniques suffer from the necessity of lengthy incubations,repeated centrifugation or use of radioactive materials.

The use of monoclonal antibodies as reagents for assaying for thepresence of apo B-100 in human body fluid samples is attractive becauseonce obtained, such reagents can be produced in relatively large amountswith consistent quality. However, there are a number of factors thatmilitate against the use of a particular monoclonal antibody as acomponent in an apo B-100 assay system.

First, the art teaches that a monoclonal antibody can be tooimmunospecific to be useful because of the antigenic heterogeneity ofits target antigen. For example, the specificity of conventionalpolyclonal antibody-containing antisera depends on a consensus ofhundreds of thousands of different antibodies that bind to antigenicdeterminants covering most or all of an antigenic protein. As a result,small changes in the structure of the antigen due to geneticpolymorphism, heterogeneity of glycosylation or slight denaturation willusually have little effect on polyclonal antibody binding. Similarly, alarger or smaller subset of antibodies from polyclonal antisera willusually bind antigens that have been modified or denatured.

In contrast, monoclonal antibodies usually bind to one antigenicdeterminant (epitope) on the antigen molecule. If, for any reason, thatdeterminant is altered, the antibody may or may not continue to bind.Whether this is a problem or an advantage depends on the individualcircumstances. If, as in the present case, the monoclonal antibody is tobe used in a diagnostic assay for an apoprotein, a minor antigenicvariation in that protein could cause gross errors.

The antigenic heterogeneity of apoprotein B-100 is well documented. Forinstance, epitope expression on apo B has been found to be modulated by(1) the composition of the associated lipids, (2) temperature of theimmunoreaction, (3) the degree of isolation of LDL from its nativeenvironment, and (4) genetic expression between individuals.

Second, because of their unique specificity, the successful use of amonoclonal antibody (MoAb) is often dependent on its affinity for thetarget antigen. For instance, whereas a MoAb may have sufficientaffinity to be useful in binding liquid and solid phase antigen whilethe MoAb is itself in liquid phase, that same antibody may not be usefulas a solid phase-affixed antibody that is useful in binding to and"pulling" the antigen out of solution.

The above problems are generic to the use of monoclonal antibodies.Those skilled in the art have therefore recognized that it is essentialto test and characterize monoclonal antibodies in any assay system inwhich they are to be used. See Goding, James W., Monoclonal Antibodies:"Principles and Practice." Pages 40-46, Academic Press, New York (1983).

SUMMARY OF THE INVENTION

One aspect this invention contemplates the hybridoma designatedHL130C2.3C5 that has ATCC accession number HB 8746. This hybridoma andits receptor molecules are also referred to herein as MB47.

In another aspect, this invention contemplates receptor molecules thatimmunoreact with apoprotein B-100 and are secreted by hybridoma ATCC HB8746.

In still another aspect, this invention contemplates the hybridomadesignated V82A6.1G4 that has ATCC accession number HB 8742. Thishybridoma and its receptor molecules are also referred to herein asMB24.

Yet another aspect, this invention contemplates receptor molecules thatimmunoreact with apoprotein B-100 and are secreted by hybridoma ATCC HB8742.

Another aspect of this invention contemplates a cell culture comprising(a) a hybridoma of this invention; (b) receptor molecules that aresecreted by said hybridoma that immunoreact with apoprotein B-100; and(c) a culture medium for the hybridoma.

A further aspect of this invention contemplates a method for assaying abody fluid sample for the presence of apoprotein B-100 comprising thesteps of:

(a) providing a body fluid sample to be assayed;

(b) providing receptor molecules in biologically active form that (i)immunoreact with apoprotein B-100, and (ii) are secreted by eitherhybridoma HB 8746 or hybridoma HB 8742;

(c) admixing the body fluid sample with the receptor molecules;

(d) maintaining the admixture under biological assay conditions for apredetermined time period sufficient for the receptor molecules toimmunologically bind apoprotein B-100 present in the sample to form animmunoreactant; and

(e) assaying the amount of immunoreactant formed.

In another aspect, this invention contemplates a method for assaying abody fluid sample for apoprotein B-100 comprising the steps of:

(a) providing a body fluid sample to be assayed;

(b) providing a solid support comprised of a solid matrix having affixedthereto in biologically active form a first receptor that immunoreactswith apoprotein B-100 and is secreted by the hybridoma having ATCCaccession number HB 8746;

(c) providing a biologically active second receptor that immunoreactswith apoprotein B-100 and is secreted by the hybridoma having ATCCaccession number HB 8742, said second receptor linked to an enzyme-labelcapable of signaling the presence of said second receptor in animmunoreactant;

(d) substantially simultaneously admixing:

(i) the body sample;

(ii) the first receptor; and

(iii) the labeled second receptor to

form a solid/liquid phase immunoreaction admixture;

(e) maintaining the admixture under biological assay conditions for apredetermined period of time period sufficient for the first receptorand the labeled second receptor to immunologically bind apoprotein B-100present in the sample to form a solid phase sandwich immunoreactant;

(f) separating said solid phase sandwich immunoreactant from the liquidphase; and

(g) assaying the amount of labeled second receptor bound in the solidphase sandwich immunoreactant that formed.

In another aspect, this invention contemplates a competitive method forassaying a body fluid sample for apoprotein B-100 comprising the stepsof:

(a) providing a body fluid sample to be assayed;

(b) providing a solid support comprised of a solid matrix having apredetermined amount of reagent apoprotein B-100 affixed thereto;

(c) substantially simultaneously admixing

(i) the body sample;

(ii) the solid support; and

(iii) a predetermined amount of

receptor molecules that immunoreact with apoprotein B-100 secreted fromeither the hybridoma having ATCC accession number HB 8746 or thehybridoma having ATCC accession number HB 8742 to form a solid/liquidphase admixture;

(d) maintaining the admixture under biological assay conditions for atime period sufficient for the receptor molecules to immunologicallybind to apoprotein B-100 molecules of the solid support and apoproteinB-100 molecules present in the body fluid sample and form a solid phaseimmunoreactant and a liquid phase immunoreactant;

(e) separating the solid phase immunoreactant from said liquid phase;

(f) admixing the solid phase immunoreactant with a biologically activesecond receptor that immunoreacts with the first receptor to form asecond solid/liquid phase immunoreaction admixture, the second receptorlinked to an enzyme-label capable of signaling the presence of thesecond receptor in an immunoreactant;

(g) maintaining said second admixture under biological assay conditionsfor a time period sufficient for the labeled second receptor toimmunologically bind to first receptor present as solid phaseimmunoreactant to form a solid phase sandwich immunoreactant;

(h) separating the solid phase sandwich immunoreactant from the liquidphase; and

(i) assaying the amount of labeled second receptor bound in said solidphase sandwich immunoreactant.

In another aspect, this invention contemplates a cell culturecomprising:

(a) the hybridoma having ATCC accession number HB 8746;

(b) receptor molecules secreted by the hybridoma that immunoreact withapoprotein B-100; and

(c) a culture medium for the hybridoma.

In another aspect, this invention contemplates a composition comprising:

(a) the hybridoma having the ATCC accession number HB 8742;

(b) receptor molecules secreted by the hybridoma that immunoreacts withapoprotein B-100; and

(c) a culture medium for the hybridoma.

In another aspect, this invention contemplates a diagnostic system forassaying for the amount of apo B-100 in a body sample comprising:

(a) a biologically active first specific binding agent that comprisesreceptor molecules that (i) immunoreact with apoprotein B-100; and (ii)are either the receptor molecules secreted by hybridoma ATCC HB 8746 orthe receptor molecules secreted by hybridoma ATCC HB 8742; and

(b) a biologically active labeled second specific binding agent forsignaling the immunoreaction of the the first binding agent with apoB-100.

A solid matrix affinity sorbant comprised of a solid phase matrixaffixed to biologically active receptor molecules that immunoreact withapoprotein B-100 and are selected from the group consisting of:

(a) the receptor molecules secreted by hybridoma HB 8746;

(b) the receptor molecules secreted by hybridoma ATCC HB 8742; and

(c) a mixture of the receptor molecules from (a) and (b).

The present invention provides several benefits and advantages.

One benefit of the present invention is that the hybridomas of thepresent invention can be used to produce in relatively large amountswith consistent quality receptors that immunoreact with apo B-100.

Another benefit of the present invention is that the receptors of thisinvention are useful, inter alia, for assaying for the amount ofcholesterol carrying apo B-100 in a body fluid sample.

One advantage of the present invention is that the receptors of thisinvention can be used in enzyme linked immunosorbant assays for apoB-100 in formats that do not require centrifugation procedures.

Another advantage is that the assay methods of this invention can becompleted in relatively short time periods.

Other advantages and benefits of the present invention will becomereadily apparent to those skilled in the art from the followingdescription of the invention, the drawings and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D and 1E are photographs of the lanes of a WesternBlot assay autoradiograph wherein the ability of MB47 and MB24 receptormolecules to immunoreact with apo B-100 and apo B-48 obtained fromdelipidated chylomicrons and VLDL is demonstrated. VLDL and chylomicronswere delipidated and subjected to SDS-polyacrylamide gel electrophoresis(SDS-PAGE) using 3-6 percent gradient gels. Sixty micrograms (ug) ofVLDL protein and 20 ug of chylomicron protein were run on alternatelanes of the gel thereby separating the proteins of each preparationaccording to size. Visualization of the electrophoretic pattern ofprotein bands by staining the gel with 0.1 percent Coomassie Bluerevealed apo B-100 and apo B-48 bands in chylomicrons and VLDL. Theprotein bands were then affixed by electrophoretic transfer tonitrocellulose paper to form a solid support. The solid support-affixedapoprotein antigens were then separately immunoreacted withimmunopurified MB47 and MB24 receptor molecules. Monoclonal MB47 andmonoclonal MB24 receptor molecules immunologically bound to solidphase-affixed antigen were detected using ¹²⁵ I-labeled goat anti-mouseIg and autoradiography.

FIG. 1A illustrates that monoclonal MB24 immunoreacts with apo B-100 andapo B-48 from VLDL (V) and chylomicrons (C). FIG. 1B illustratesmonoclonal that MB47 immunoreacts with apo B-100 from V and C but notwith apo B-48 from either V or C. FIG. 1C is a negative control showingthat a monoclonal antibody specific for sheep red blood cells does notimmunoreact with any antigen present. FIG. 1D is another negativecontrol showing that an immunopurified polyclonal antiserum tophenyl-beta-O-glucoside does not recognize any antigens present. FIG. 1Eis a positive control showing that an immunopurified rabbit polyclonalantiserum against human LDL-apoprotein recognizes both apo B-100 and apoB-48 in both V and C.

FIG. 2 is a graph showing the percentage of ¹²⁵ I-labeled LDL particlesbound (oridinate) by increasing molar concentrations of monoclonalantibody MB47 [abscissa; Ab concentration (MoAb)] in a fluid phaseradioimmunoassay (RIA).

LDL was prepared from pooled plasma of 10 subjects or from onenormolipidemic subject . Plasma was obtained by plasmapharesis ofsubjects following an approximately twelve-hour fasting period.

FIG. 3 is a bar graph showing the degree of inhibition by antibody MB47(open bars: MB47) and excess unlabeled human LDL (hatched bars: LDL) of¹²⁵ I-human LDL binding, internalization and degradation by culturedhuman fibrobalsts. Fibroblast monolayers were grown in 35 millimeterwells in DME containing 10 percent fetal calf serum.

Fibroblast LDL-receptors were stimulated by approximately 24 hours ofpreincubation of the fibroblasts with growth medium containing 2.5milligrams per milliliter (mg/ml) lipoprotein-depleted serum (LDS)(DME-LDS). DME-LDS containing 2.5 micrograms per milliliter (ug/ml) ¹²⁵I-LDL and either 20 percent MB47 hybridoma culture supernatant (v/v) ora 200-fold excess of unlabeled LDL (final concentration, 500 ug/ml) wereadmixed and maintained (incubated) for about 16 hours at 4 degrees C.prior to being placed on the fibroblast monolayers.

Determination of binding, internalization, and degradation wereperformed in triplicate, and are expressed as a percentage of controlvalues that were determined in the absence of monoclonal receptor MB47.Inhibition of specific binding, internalization, and degradation bymonoclonal receptor MB47 was comparable to that produced by a 200-foldexcess of unlabeled LDL.

FIGS. 4A and 4B are graphs showing the ability of monovalent Fabfragments of monoclonal antibody MB47 to inhibit ¹²⁵ I-human LDL bindingand degradation by human fibroblasts. Individual media containingincreasing amounts of MB47-Fab fragments and a constant amount of ¹²⁵I-LDL (2.5 um/ml) were admixed and maintained (incubated) for a timeperiod of about 15 hours at 4 degrees C. prior to being placed on thefibroblast monolayers. The Fab concentration is expressed as the molarratio of Fab/LDL present in each medium (assuming a molecular weight(MW) of Fab to be 40,000 daltons and a molecular weight of apo B to be550,000 daltons).

Binding and degradation are expressed as the percentage of controlvalues in the absence of MB47-Fab fragments. All determinations wereperformed in duplicate. Excess unlabeled LDL (final concentration, 500ug/ml) produced greater than 95 percent inhibition of ¹²⁵ I-LDL binding(FIG. 4A) and degradation top graph (FIG. 4B). Similar results wereobtained in three studies with different Fab preparations of monoclonalreceptor MB47.

FIG. 5A illustrates the ability of a known, constant amount of horseradish peroxidase-labeled MB47 (HRPO-MB47) receptors to immunoreact withsolid phase affixed reagent apo B-100 in the presence of increasingamounts of MB24 receptor molecules. The ordinate is in relative opticaldensity units while the abscissa is in units of micrograms permilliliter of unlabeled antibody protein added as (ug/ml) competitor.

A constant amount (20 ug) of HRPO-coupled MB47 receptors wassubstantially simultaneously admixed with increasing amounts ofunlabeled MB47 or unlabeled MB24 receptors and solid phase-affixedreagent apo B-100 (LDL). The admixtures were maintained for 3 hours at25 degrees C. thereby allowing the receptors to immunologically bind thereagent apo B-100 and form a solid phase immunoreactant. The amount ofsolid phase-bound labeled MB47 was then assayed as in the competitionELISA described in the Materials and Methods section.

FIG. 5A illustrates that the presence of increasing amounts of unlabeledMB47 receptors in the immunoreaction admixture correspondingly decreasesthe amount of labeled MB47 receptors bound as solid phaseimmunoreactant. Thus, unlabeled MB47 competes with labeled MB47 for LDL.

On the other hand, FIG. 5A also illustrates that increasing amounts ofunlabeled MB24 receptors do not significantly decrease the amount oflabeled MB47 bound as solid phase immunoreactant. Thus, unlabeled MB24does not compete with labeled MB47 for binding to LDL.

FIG. 5B illustrates that similar results are obtained using HRPO-labeledMB24 receptors and unlabeled MB47 receptors. MB47 and MB24 receptorstherefore bind to different epitopes that are sufficiently separated onthe surface of apo B-100 so as to allow binding of both receptors to asingle apo B-100 molecule without sterically competing with andinhibiting binding.

FIG. 6A represents the binding of ¹²⁵ I-labeled B-47 to LDL in a fluidphase RIA. The ordinate is in units of femtomoles (fmols) bound whereasthe abscissa is in units of nanomoles (nM) of the antibody admixed.

Immunopurified monoclonal antibody MB47 was iodinated with ¹²⁵ I usingthe Iodogen technique to a specific activity of 3000 counts per minuteper nanogram (cpm/ng). Following extensive dialysis againstphosphate-buffered saline (PBS), over 95 percent of the radioactivitywas precipitable by 10 percent trichloroacetic acid (TCA). Greater than98 percent of the ¹²⁵ I-MB47 bound to a LDL column. Assays wereperformed in triplicate in 10×75 milliliter (mm) silicone-coated glasstubes. Increasing concentrations of ¹²⁵ I-MB47 in 0.1 ml of bovine serumalbumin-barbital (BSA-barbital) buffer (pH value 8.0) were added to 100ng of pooled, normolipidemic human LDL diluted in 0.2 ml of BSA-barbitalbuffer. Each tube contained 182 fmoles of LDL apo B (assuming an apo Bmolecular weight of 550,000 daltons).

After incubation (admixture and maintenance) for a time period of 16hours at 4 degrees C., LDL was quantitatively precipitated by alipoprotein-depleted rabbit antiserum specific for human LDL. [Only thefraction of the rabbit antiserium having a density (d) greater than 1.21g/ml was used because monoclonal antibody MB47 binds rabbitapolipoprotein B.]

Preliminary studies established a concentration of delipidated rabbitantiserum that precipitated greater than 98 percent of 100 ng of ¹²⁵I-human LDL. After addition of the rabbit antiserum, the tubes wereincubated for about 16 hours at 4 degrees C.

The supernatants were removed, and the pellets were washed twice with 2ml of ice cold barbital buffer (pH value 8.0). Nonspecific binding andprecipitation were determined in two sets of parallel tubes. In thefirst set, no human LDL was added to the initial incubation, but thesame amount of rabbit second antibody was added. In the second set oftubes, nonimmune rabbit serum (d greater than 1.21 mg/ml fraction) wassubstituted for the immune rabbit serum antibody.

Both methods yielded substantially identical values for nonspecificbinding, which was linear with increasing concentrations of ¹²⁵ I-MB47monoclonal antibody, and in all cases was less than 1 percent of thetotal counts added. Specific ¹²⁵ I-MB47 binding to LDL was obtained bysubtracting nonspecific binding from total binding. Binding data wereanalyzed utilizing a linear regression program for Scatchard analysis ofligand binding systems, which provided an estimate of the antibodyaffinity constant (Ka) and the receptor or epitope concentration.

The Ka of ¹²⁵ I-MB47 for LDL was thereby determined to be 3.82×10⁹ M⁻¹.Extrapolation of the line to conditions of infinite antibody excessyielded an estimate of 212 fmols (35 ng) of ¹²⁵ I-MB47 bound by 182fmoles (100ng) of LDL when the molecular weight of apo B is assumed tobe 550,000 daltons. These data are shown in FIG. 6B wherein the ordinateis in units of the ratio of bound (B) to free (F) (B/F) antibody,whereas the abscissa is in units of fmoles of bound antibody (fmols ofMB47).

FIG. 7 illustrates the correlation between the mg of LDL-cholesterol perdeciliter (dl) of sample determined by Lipid Research Clinic Procedures,HEW Publication No. 75-628 (NIH), 2nd ed., Wash., D.C. Gov. Print. Off.(1974), and mg of apo B-100 per dl of sample determined using thenon-competitive ELISA described in the materials and methods section.

The correlation coefficient, r=0.89, determined by the Spearman RankCorrelation test for non-parametric data [Sokal et al., Biometry, 2nded., W.H. Freeman Co., San Fransicso, CA, 561-616 (1981)], indicates asignificant correlation between the levels of apo B-100 determined bythe non-competitive ELISA described herein and the LDL-cholesterollevels in 60 human plasma sampels.

FIG. 8 is similar to FIG. 7 and illustrates a significant correlation,r=0.92, between the levels of apo B-100 as determined by a competitionELISA method of this invention and the LDL-cholesterol levels in thesame 60 human samples.

FIG. 9 illustrates the significant correlation, r=0.92, as determined bythe Spearman Rank Correlation test, between the results obtained usingthe non-competitive ELISA (ordinate) and the competitive ELISA(abscissa) on the same 60 human samples used in FIGS. 7 and 8.

DETAILED DESCRIPTION OF THE INVENTION I. General Discussion A.Definitions

The term "antibody" refers to a receptor molecule that is a member of afamily of glycosylated proteins called immunoglobulins, which canspecifically combine with an antigen.

An "antibody combining site" is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen.

The word "antigen" has been used historically to designate an entitythat is bound by an antibody, and also to designate the entity thatinduces theproduction of the antibody. More current usage limits themeaning of antigen to that entity bound by an antibody, while the"immunogen" is usedfor the entity that induces antibody production.Where an entity discussed herein is both immunogenic and antigenic, itwill generally be termed an antigen.

"Antigenic determinant" refers to the actual structural portion of theantigen that is immunologically bound by an antibody combining site. Theterm is also used interchangeably with "epitope".

The term "biologically active" refers at least to the ability tospecifically bind ligand or specific binding agent although othergeneral or effector capability can also be present.

The word "complex" as used herein refers to the product formed when aspecific binding agent binds to a target ligand. Exemplary complexes areimmunoreactants, protein A bound to an antibody and the like.

"ELISA" refers to an enzyme-linked immunosorbent assay that employs anantibody or antigen bound to a solid phase and an enzyme-antigen orenzyme-antibody conjugate to detect and quantify the amount of antigenor antibody present in a sample. A description of the ELISA technique isfound in Chapter 22 of the 4th Edition of Basic and Clinical ImmunologybyD.P. Sites et al., published by Lange Medical Publications of LosAltos, CAin 1982 and in U.S. Pat. Nos. 3,654,090; 3,850,752; and4,016,043, which are all incorporated herein by reference.

"Enzyme" refers to a protein capable of accelerating or producing bycatalytic action some change in a substrate for which it is oftenspecific.

"Epitope" refers to that portion of a molecule that is specificallyrecognized by an antibody combining site. It also is referred to as thedeterminant or antigenic determinant.

"Idiotopes" or "idiotypic determinants" are antigenic determinants onthe variable and hypervariable portions of an antibody molecule that canbe recognized by a combining site of other antibodies. Idiotopes areusually divided into two types, those that are binding site-associateddeterminants and those that are non-binding site-associated. Thecollection of idiotpes on an antibody molecule constitutes its idiotype.It is believed that an antibody combining site produced by a hybridomapossesses a single, unique set of idiotopes; i.e., a unique antibodycombining site idiotype.

"Immunoreactant" as used herein refers to the product of animmunological reaction; i.e., that entity produced when a ligand isimmunogically bound by a receptor molecule. An "immunoreactant" is aparticular type of "complex".

The word "isolated" as used herein in relation to receptor moleculesmeans that substantially only one species of antibody combining site ispresent.

The terms "labeling means", "indicating group" or "label" are usedinterchangeably herein to include single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal to indicate the presence of a immunoreactant. Any labeling meanscan be linked to or incorporated in a receptor or used separately, andthose atoms or molecules can be used alone or in conjunction withadditional reagents. Such indicating groups or labels are themselveswell-known in immunochemistry and constitute a part of this inventiononlyinsofar as they are utilized with otherwise novel receptors, methodsand/orsystems.

"Ligand" refers to a molecule that contains a structural portion that isbound by a specific receptor, e.g., an antigen that is bound by areceptor.

The term "receptor" is used herein to indicate a biologically activemolecule that immunologically binds to (or with) an antigen. Suchbinding typically occurs with an affinity of about 10⁵ to about 10¹⁰liters per mole (M⁻¹) and is a specific interaction of the epitope oftheantigen with the antibody combining site of the receptor.

A receptor molecule of the present invention is any intact antibody,substantially intact antibody or an antibody combining siteidiotype-containing polypeptide portion of an antibody (e.g., an Fabfragment) such as in ascites fluid or tissue culture supernatant.

The word "receptor" is also used herein for molecles on cell surfacesthat bind other molecules. Cell surfaces receptors are alwaysdenominated herein with the name of the bound entity preceding the word"receptor" to avoid any ambiguity. An exemplary cell surface "receptor"is the previously described LDL receptor.

Biological activity of a receptor molecule is evidenced by theimmunologic reaction of the receptor with its antigenic ligand upontheir admixture inan aqueous medium to form an immunoreactant, at leastat physiological pH values and ionic strengths. Preferably, biologicalactivity occurs under biological assay conditions; i.e., thoseconditions wherein the receptor molecules of this invention bind to theantigenic ligand within a pH valuerange of about 5 to about 9, at ionicstrengths such as that of distilled water to that of about one molarsodium chloride, and at temperatures of about 4 degrees C. to about 45degrees C. All of the receptor molecules described herein werebiologically active.

Antibody combining site idiotype-containing polypeptide portions(antibody combining sites) of antibodies are those portions of antibodymolecules that contain the combining site idiotopes and bind to theligand, and include the Fab, Fab', F(ab')₂ and F(v) portions of theantibodies. Fab and F(ab')₂ portions of antibodies are well known in theart, andare prepared by the proteolytic reaction of papain and pepsin,respectively, on substantially intact antibodies by methods that arewell known. See for example, U.S. Pat. No. 4,342,566 to Theofilopolousand Dixon. Fab' antibody portions are also well known and are producedfrom F(ab')₂ portions followed by reduction of the disulfide bondslinkingthe two heavy chain portions as with mercaptoethanol, and thenalkylation of the resulting protein mercaptan with reagent such asiodoacetamide. Intact antibodies are preferred, and along with Fabportions are utilized as illustrative of the monoclonal receptormolecules this invention.

The words "secrete" and "produce" are often used interchangeably in theartas to cells from which antibody molecules are obtained. Cells thatproduce antibodies may, however, not secrete those molecules into theirenvironment. The hybridoma cells of interest herein secrete monoclonalantibodies into their environment. Nevertheless, such cells are oftenreferred to herein as "antibody-producing" cells, and their antibodiesarereferred to as being "produced" in keeping with the phrase utilizedin the art.

The term "specific binding agent" as used herein refers to a molecularentity capable of selectively binding a ligand. Exemplary specificbindingagents are receptors, complement fragments, protein A and thelike.

The phrase "substantially pure" as used herein in relation to receptormolecules means that, within detectable limits, only one species ofantibody combining site is present as an effective binding agent for apoB-100. Thus, while a substantially pure receptor molecule preparationcan contain more than one species of antibody combining site, such apreparation displays a single binding affinity for apo B-100. Forinstance, tissue culture supernatants produced by a hybridoma of thisinvention typically contain myeloma proteins as well as receptors ofthis invention. A receptor molecule in substantially pure form istypically designated a "monoclonal antibody" by those skilled in the artbecause such compositions are produced using monoclonal hybridomacultures.

The phrase "substantially simultaneously" as used herein in relation totheadmixture of 3 or more antigen and receptor components to form animmunoreaction admixture means that all components are present andadmixedin a single admixture within about 15 minutes and preferablywithin about 5minutes of the admixture of any 2 of the components.

B. Hybridomas and Monoclonal Receptors

The present invention contemplates a hybridoma, having the laboratorydesignation HL130C2.3C5, that produces receptor molecules that:

(a) immunoreact with a conserved antigenic determinant on apoproteinB-100;

(b) competitively inhibit the binding of apoprotein B-100 to LDLreceptor; and

(c) have an affinity constant for LDL of about 3.82×10⁹ M⁻¹in a fluidphase competitive equilibrium radioimmunoassay (RIA). These receptormolecules are usually referred to as MB47.

The present invention also contemplates a hybridoma, having thelaboratory designation V82A6.1G4, that produces receptor molecules thatimmunoreact with an apoprotein B-100 antigenic determinant and have anaffinity constant for LDL of about 3.0×10⁹ M⁻¹ in a solid phasecompetitive equilibrium RIA. These receptor molecules are usuallyreferredto as MB24.

Hybridomas HL130C2.3C5 and V82A6.1G4 were deposited with the AmericanType Culture Collection (ATCC), Rockville, MD on Mar. 6, 1985 under thefollowing ATCC accession numbers:

    ______________________________________                                                     Receptor                                                         Hybridoma    Designation                                                                             ATCC Accession No.                                     ______________________________________                                        V82A6.1G4    MB24      HB 8742                                                HL130C2.3C5  MB47      HB 8746                                                ______________________________________                                    

The above ATCC deposits were made in accordance with the Budapest Treatyonthe International Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure.

The hybridomas of the present invention were formed by fusing anantibody-producing cell and a myeloma cell line. Such receptor producingcells were first described by Kohler and Milstein, Nature, 256, 495(1975), which description is incorporated herein by reference. Receptorsare typically obtained from the supernatants of hybridoma cell cultures,preferably monoclonal cell cultures, or, alternatively, from ascitesfluidor other body fluids obtained from non-human, warm-blooded hostanimals, preferably those that are histocompatible or immunocompromised,into whichthe hybridoma cells were introduced and cultured.

Thus, in another embodiment, the present invention contemplates a cellculture comprising (a) a hybridoma of this invention; (b) receptormolecules that are secreted by the hybridoma that immunoreact withapoprotein B-100; and (c) a culture medium for the hybridoma. Mediausefulfor the preparation of these compositions are both well known inthe art and commercially available and include synthetic culture media,inbred mice and the like. An exemplary synthetic medium is Dulbecco'sminimal essential medium (DMEM; Dulbecco et al., Virol. 8, 396 (1959))supplemented with 4.5 gm/l glucose, 20 mm glutamine, and 20 percentfetal calf serum. An exemplary inbred mouse strain is the Balb/c.

In still another embodiment, the present invention contemplates thereceptors, designated MB47 and MB24, that are produced by the hybridomasdesignated HB 8746 and HB 8742 respectively, and immunoreact withapoprotein B-100. Thus, a receptor of this invention can be prepared byculturing in a suitable medium an appropriate hybridoma of thisinvention and recovering the receptor from the medium.

Previously, Curtiss et al., J. Biol. Chem., 257, 15213 (1982), reportedproduction and characterization of 11 apo B specific receptor molecules,including that designated MB24 produced by hybridoma HB 8742. HybridomaHB8742 was obtained, as described in more detail in the Materials andMethods section, by fusing splenocytes of mice immunized with humanVLDL.

The IgG fraction of MB24 containing ascites fluid generates fromintraperitoneal growth of HB 8742 was characterized by isoelectricfocusing (IEF). As noted in Curtiss et al., supra, the fusion wasperformed with P3×63Ag8 myeloma cells that secrete an IgG₁ kimmunoglobulin. Therefore, upon IEF, HB 8742 ascites fluid demonstrateda unique pattern of multiple protein bands representing randomly mixedheavyand light chain-containing immunoglobulin molecules in addition tothe P3×63Ag8 myeloma IgG₁ k antibody and receptor MB24.

Hybridoma HB 8746 produces MB47 receptor molecules and was formed byfusingsplenocytes of mice immunized with LDL and P3×63Ag8,653.1 myelomacells. This variety of the parent myeloma does not secrete a myelomaprotein. IEF of HB 8746 ascites fluid reveals a unique pattern ofprotein bands representing the IgG2a heavy and kappa light chains. Thus,both hybridomas of this invention can be characterized in part by theIEF pattern of the receptor molecules they produce.

While the V82A6.1G4 hybridoma of this invention produces more than onetypeof receptor molecule, the receptor molecules of this invention canbe easily identified and isolated by their individual abilities toimmunoreact with apo B-100 antigenic determinants. The antigenicspecificities of MB24 and MB47 were examined by assaying theirindividual abilities to immunoreact with apoproteins obtained fromchylomicrons, VLDL, LDL and HDL in the Western blot assay describedhereinbelow.

The data so obtained indicated that MB47 and MB24 immunoreact with apoB-100 obtained from LDL, VLDL and chylomicrons, but not with apo B-48fromVLDL or chylomicrons. The ability of both MB47 and MB24 toindividually immunoreact with apo B-100 from chylomicrons and VLDL isshown in FIGS. 1A, 1B, 1C, 1D and 1E.

1. Characterization of the Apo B-100 Antigenic DeterminantImmunologically Bound By MB47

Previous studies have demonstrated antigenic heterogeneity in apo B-100.That is, some apo B-100 epitopes are not expressed by all LDL particles.Thus, admixture with an excess of certain monoclonal antibodies in afluidphase RIA does not result in immunological binding of allradiolabeled LDL (¹²⁵ I-LDL) particles.

To determine whether the epitope recognized by MB47 receptor moleculeswas uniformly expressed by all LDL, the ability of MB47 toimmunologically bind to ¹²⁵ I-LDL in a fluid phase RIA was studied. LDLisolated frompooled plasma of 10 normal subjects and from a singlenormal subject were radiolabeled as described hereinbelow and wereadmixed with biologically active MB47 receptor molecules to form animmunoreaction admixture. The admixture was maintained under biologicalassay conditions for a predetermined time period sufficient for the MB47receptor molecules to immunologically bind to apo B in each sample andform an immunoreaction product (immunoreactant).

The maximal amount of ¹²⁵ I-LDL bound by an excess of MB47 receptormolecules was assayed by precipitation of all receptor molecules withIgSORB (The Enzyme Co., Boston, MA) and quantitation of ¹²⁵ I-LDLassociated counts in the precipitate in a gamma counter. The results,expressed as a percentage of ¹²⁵ I-LDL precipitated by trichloroaceticacid (TCA) and shown in FIG. 2, demonstrate that essentially all ¹²⁵I-LDL was bound by antibody, indicating that the epitope recognized andbound by MB47 is expressed by all LDL particles.

2. Competitive Inhibition of the Binding of Apo B-100 to LDL--Receptorby MB47

Apoprotein B-100 is the major apoprotein in human and other mammalianLDL, and it mediates binding of LDL to the fibroblast LDL receptor.Previous studies have delineated the central role of the LDL receptor inmammalian lipoprotein metabolism. The fact that LDL particles isolatedfrom multipleanimal species bind specifically to the human LDLreceptor-binding domain, the binding site on the apo B-100 molecule mustbe evolutionarily conserved and thus expressed by LDL particles from allanimal species.

To examine whether MB47 receptor molecules immunoreact with an antigenicdeterminant located within the LDL receptor binding domain of human apoB-100, the ability of MB47 to inhibit binding of ¹²⁵ I-LDL to thecellular LDL receptor was examined. This was accomplished by admixingMB47receptor molecules, in the form of whole antibody molecules, with¹²⁵ I-LDL to form an immunoreaction admixture. The immunoreactionadmixture was then maintained under biological assay conditions for apredetermined time period sufficient for the MB47 receptor molecules toimmunogically bind the ¹²⁵ I-LDL present and form an immunoreactant.

Subsequently, the immunoreactant-containing immunoreaction admixture waslayered over human fibroblasts that express fibroblast LDL receptors.The cultures were then maintained for a predetermined time periodsufficient for the fibroblast LDL receptors to specifically bind any LDLreceptor-binding sites available on the ¹²⁵ I-LDL-MB47immunoreactionproduct. It is presumed that such cellular receptor-LDLinteraction is inhibited if receptor molecules as exemplified by MB47immunoreact with anapo B-100 antigenic determinant whose structure isalso involved in LDL-receptor binding.

The results of this study, shown in FIG. 3, indicate that MB47 receptormolecules inhibited cellular receptor-mediated binding, internalization,and degradation of human ¹²⁵ I-LDL by human fibroblasts to an extentcomparable to that produced by a 200-fold excess of unlabeled LDL.

To examine the possibility that MB47 receptors bind to an epitopeadjacent to the apo B receptor domain, and because of their size,sterically hinderthe binding of apo B to the LDL receptor, the capacityof Fab fragments of MB47 antibody molecules to inhibit human LDL uptakeand degradation was also studied in the above described fibroblastassay.

As shown in FIGS. 4A and 4B, MB47-Fab fragments significantly blockedspecific cellular binding and degradation of LDL. Because MB47 Fabfragments are substantially smaller than intact MB47-antibody moleculesitis believed that the epitope recognized by the MB47 antibody combiningsiteis contained within the LDL receptor binding domain on LDL apoB-100. Antibody MB24 does not have the properties of antibody MB47 anddoes not inhibit the binding and degradation of ¹²⁵ I-LDL by culturedfibroblasts.

3. Steric Inhibition

For some embodiments of the assay method of this invention the first andsecond receptors must bind to different epitopes of the apo B-100molecule, and those epitopes must be sufficiently separated such thatthe binding of one receptor does not sterically inhibit the binding ofthe other receptor. The ability of MB47 and MB24 to competitivelyinhibit the immunological binding of each other to solid phase-affixedreagent apo B-100 was therefore examined.

The results of that study, shown in FIGS. 5A and 5B, indicate that a 70fold excess of unlabeled MB24 did not significantly inhibitperoxidase-labeled MB47 from binding to reagent apo B-100. Similarly, a70-fold excess of unlabeled MB47 did not significantly inhibitperoxidase-labeled MB24 from binding to reagent apo B-100. Thus, MB24and MB47 bind to different epitopes on apo B-100 and those epitopes aresufficiently separated such that MB24 and MB47 as intact antibodies donotinhibit the binding of each other to a single apo B-100 molecule.

4. Stoichiometry and Affinity of MB47 Binding to Apo B-100

To determine the number of antigenic determinant sites per apo B-100molecule on LDL that were recognized by MB47 receptor molecules, anantibody-labeled RIA was used. In this assay, MB47 receptor moleculeswerepurified (isolated) from ascites fluid, and were radiolabeled (¹²⁵I-MB47) by well known methods that are described in more detail in theMethods and Materials section.

As shown in FIG. 6A, increasing amounts of ¹²⁵ I-MB47 were admixed witha fixed amount of apo B-100 in the form of LDL in separate reactionadmixtures. The admixtures were maintained under biological assayconditions for a predetermined time period sufficient for the ¹²⁵ I-MB47receptor molecules to immunologically bind to the apo B-100 (LDL) andform an immunoreactant. The presence of immunoreactant was then assayedby quantitatively precipitating the LDL (bound and free) using a rabbitantiserum specific for human LDL, and detecting the amount of ¹²⁵ I-MB47present as immunoreactant by gamma counting.

The specific immunological binding of ¹²⁵ I-MB47 receptor molecules wassaturable as shown in FIG. 6A. Furthermore, a Scatchard plot of thebinding data obtained in these studies was linear (FIG. 6B), suggestinguniformity of MB47 binding sites on LDL particles.

In addition, the apparent affinity constant (Ka) of MB47 for human apoB-100 in the form of LDL as assessed by the antibody-labeled RIA wasfoundto be 3.82×10⁹ M⁻¹ Scatchard analysis also revealed that a maximumof 212 fmoles of ¹²⁵ I-MB47 antibody bound to 182 fmoles of LDL,indicating that only one MB47 molecule binds to each molecule of apoB-100. That is, MB47 binds to a single, unique antigenic determinant onapo B-100.

The average affinity constant of MB47 for apo B-100 was also assessed inthe before described antigenlabeled RIA. In this competitive equilibriumfluid phase RIA, unlabeled apo B-100 present as LDL produced fulldisplacement of ¹²⁵ I-LDL. The calculated affinity constant of MB47receptor molecules present as whole, intact antibody for LDL in thisassaywas 4×10⁹ M⁻¹, which was in good agreement with the Ka determinedby the antibody-labeled assay described hereinbefore.

C. Assay Methods

The receptor molecules of the present invention are particularly usefulforassaying the presence and amount of apoprotein B-100 in a body fluidsamplesuch as blood, serum or plasma.

In one embodiment, the present invention contemplates a method forassayinga body sample for the amount of apoprotein B-100 comprising thefollowing steps:

(a) Providing a body sample to be assayed. Typically such a sample isprovided as a measured quantity or known amount of blood and morepreferably as plasma or serum. Methods for providing samples of blood,plasma and serum are well known in the art and will not be discussedfurther herein.

(b) Providing receptor molecules in biologically active form that (i)immunoreact with apoprotein B-100 and (ii) are secreted by either thehybridoma having the ATCC accession number HB 8746 or the hybridomahavingthe ATCC accession number HB 8742, and present in an amounteffective for carrying out the assay.

In preferred embodiments the receptor is an intact antibody or a Fabfragment.

The effective amount of receptor molecules can differ, inter alia, withtheparticular assay method utilized as is well known. Also well known isthe ease with which the effective amount can be determined usingstandard laboratory techniques by one skilled in the art.

(c) Admixing the body fluid sample with the receptor molecules of step(b) to form an immunoreaction admixture.

(d) The admixture is maintained under biological assay conditions for apredetermined time period from minutes to hours such as about 10 minutesto biological assay conditions for a predetermined time about 16-20hours that is sufficient for the antibody combining sites of thereceptor molecules to immunologically bind apoprotein B-100 in the bodysample and form an immunoreactant (first complex). Biological assayconditions are those that maintain the biological activity of thereceptor molecules of this invention and include a temperature range ofabout 4 degrees C. to about 45 degrees C., a pH value range of about 5to about 9 and an ionic strength varing from that of distilled water tothat of about one molar sodium chloride. Methods for optimizing suchconditions are well known in the art.

(e) Assaying the amount of any immunoreactant that formed and therebythe amount of apo B-100 present in said sample.

In preferred embodiments, the body fluid sample of step (a) is furtherprepared for assaying according to step (e) by the following steps:

(f) Providing biologically active second receptor molecules secreted bytheremaining of either hybridoma of step (b);

(g) Admixing a predetermined amount of the second receptor moleculeswith the body fluid sample to form an immunoreaction admixture.

(h) Maintaining the second receptor/body fluid sample admixture soformed in a manner similar to that described in step (d) to form asandwich immunoreactant (second complex) that contains one MB47 moleculeand one MB24 molecule immunologically bound to one apo B-100 molecule.

The above described general assay methods can be performed, as is wellknown in the art, using a variety of different formats. Thus, whereasthe more specific assay methods described hereinbelow use solid phaseformats,the invention is not so limited.

Solid phase assay formats can be performed using either receptormolecules or antigen affixed to a solid matrix to form a solid support.In those embodiments wherein the solid support contains the receptor ofstep (b), the admixture of step (c) is a solid/liquid phase admixtureand the immunoreactant of step (d) is a solid phase immunoreactantcontaining bodysample apo B-100.

In those embodiments wherein the solid support contains reagent apoB-100, the admixture of step (c) is also a solid/liquid admixture butthe body sample apo B-100-containing immunoreactant of step (d) is aliquid phase immunoreactant. Reagent apo B-100 is biologically active;i.e., antigenic,apo B-100 that is provided by a source other than thatwhich is under investigation, typically in the form of isolated LDL.

Assaying the amount of apo B-100 bound as immunoreactant in step (d) canbeaccomplished, directly or indirectly, by assay techniques well knownin theart. For example, homogeneous assay systems such as thosedescribed in U.S.Pat. Nos. 4,536,479; 4,233,401; 4,233,402 and 3,996,345which are all incorporated herein by reference, may be used.

In preferred solid phase embodiments, the body fluid is further preparedfor assaying by using a labeled specific binding agent. The type andspecificity of the labeled specific binding agent depends, as is wellknown in the art, on the method and format used.

In the preferred solid phase embodiments wherein the solid supportcontainsa receptor of step (b); i.e., a receptor of this invention, theamount of solid phase-bound apo B-100 is prepared for assaying by thefollowing steps:

(i) Providing biologically active labeled second receptor molecules thatbind to apoprotein B-100 present in the body sample to form animmunoreactant. The label of the labeled second receptor is capable ofsignaling the presence of the labeled second receptor in animmunoreactant.

In particularly preferred embodiments, the labeled second receptormolecules immunoreact with a second apo B-100 epitope that is differentfrom the epitope with which the solid phase receptor molecules react anddo not substantially inhibit the solid phase receptor molecules fromreacting with apo B-100. Preferably, the labeled second receptormoleculesare those secreted by the remaining of either hybridoma of step(b); i.e., the recited receptor molecules not chosen as first receptormolecules.

Methods for determining whether a receptor molecule will inhibit(interferewith) the immunological binding to the same antigen of anotherreceptor molecule are well known in the art and are described in moredetail hereinbelow.

(j) Admixing a predetermined amount of the labeled second receptormolecules with the body fluid sample to form an immunoreactionadmixture.

The admixture so formed can be a liquid admixture as when, step (i) isperformed prior to step (b) hereinbefore, or it can be a solid/liquidadmixture when the labeled second receptor is admixed substantiallysimultaneously with or after step (b). When step (i) is performed beforeor substantially simultaneously with step (b), the labeled secondreceptormolecules immunoreact with a second apo B-100 epitope that isdifferent from the epitope with which the solid phase receptor moleculesreact and do not substantially inhibit the solid phase-bound receptormolecules fromimmunoreacting with apo B-100.

In preferred embodiments step (i) is performed substantiallysimultaneouslywith step (b) or after step (c).

(k) The labeled second receptor/body fluid sample admixture so formed ismaintained in a manner similar to that described in step (d) to form animmunoreactant.

The solid phase-bound receptor molecules and second receptor moleculesare thus immunologically bound to apo B-100 present in the body fluidsample thereby forming a solid phase-bound sandwich immunoreactant thatcontains label bound as part thereof. That is, a solid phase sandwichimmunoreactant that contains a label is formed when one molecule of apoB-100 immunoreacts with both a solid phase-bound receptor molecule and alabeled second receptor molecule. In preferred embodiments, any labeledsecond receptor molecules that do not form a part of the solidphase-boundimmunoreactant (i.e., those not immunologically bound to apoB-100 which itself is bound to solid phase receptor molecules) areseparated from the immunoreactant, preferably by washing, prior toassaying for the amount oflabeled second receptor present asimmunoreactant.

Assaying the amount of immunoreactant formed according to step (e) isaccomplished by assaying for the amount of the labeled second receptorbound as part of the immunoreactant that contains apo B-100. Thisprovidesa direct assay for the amount of apo B-100 in the sample. Thatamount can be zero, thereby indicating no apo B-100 present in thesample, within thelimits that can be detected. Methods for assaying forthe amount of a labeled second receptor depend on the label used, suchlabels and assay methods being well known in the art.

In the preferred solid phase embodiments wherein the solid supportcontainsreagent apo B-100, the amount of immunoreactant formed in step(d) is prepared for assaying by the following steps:

(1) Admixing a biologically active labeled specific binding agent,preferably a receptor molecule, that binds to any receptor moleculesutilized in step (b) present as solid phase immunoreactant to form acomplex, preferably a second immunoreactant. The label of the labeledspecific binding agent is capable of signaling the presence of thelabeledspecific binding agent in a complex. In preferred embodiments ofthese assay types, steps (i)-(k) are carried out after step (d).

(m) Admixing a predetermined amount of the labeled specific bindingagent with the body fluid sample to form a reaction, preferablyimmunoreaction, admixture.

(n) The labeled specific binding agent/first immunoreactant admixture soformed is maintained in a manner similar to that described in step (d)to form a complex.

The solid phase complex thus formed contains a label bound as partthereof.In preferred embodiments, any labeled specific binding agent notbound as part of the solid phase complex is separated from the complex,preferably by washing, prior to assaying for the presence of solidphase-bound label.

Assaying the amount of immunoreactant formed according to step (e) isaccomplished by assaying for the amount of label present as part of thecomplex. This provides an indirect assay for the amount of apo B-100present in the sample.

The labeling of proteinaceous antigens and specific binding agents iswell known in the art. For instance, receptors produced by hybridomascan be labeled by metabolic incorporation of radioisotope-containingamino acids provided as a component in the tissue culture medium. Seefor example Galfre et al., Meth. Enzymol. 73, 3-46 (1981).

The techniques of protein conjugation or coupling through activatedfunctional groups are particularly applicable and result in label beingcovalently linked to antigen or specific binding agent. See, forexample, Aurameas, et al., Scand. J. Immunol. Vol. 8, Suppl 7, 7-23(1978) and U.S.Pat. No. 4,493,795 which is incorporated herein byreference. In addition, site-directed coupling reaction can be carriedout so that the label does not substantially interfere with thebiological activity of an antigen or receptor, for example, Rodwell etal., Biotech. 3, 889-894 (1985).

The labeling means can be a fluorescent labeling agent that chemicallybinds to antibodies or antigens without denaturing them to form afluorochrome (dye) that is a useful immunofluorescent tracer. Suitablefluorescent labeling agents are fluorochromes such as fluoresceinisocyanate (FIC), fluorescein isothiocyanate (FITC),5-dimethylamin-l-naphthalenesulfonyl chloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB 200 SC) and the like. A description ofimmunofluorescence analysis techniques is found in DeLuca,"Immunofluorescence Analysis" in Antibody As A Tool, Marchalonis et al.,eds. J. Wiley & Sons, Ltd., p. 189-231, 1982, which is incorporatedhereinby reference.

In preferred embodiments, the indicating group is an enzyme such ashorseradish peroxidase (HRPO), glucose oxidase or the like. Where theprincipal indicating group is an enzyme such as HRPO or glucose oxidase,additional reagents are required to visualize the fact that areceptor-ligand complex (immunoreactant) has formed. Such additionalreagents for HRPO include hydrogen peroxide and an oxidation dyeprecursorsuch as diaminobenzidine. An additional reagent useful withglucose oxidaseis 2,2,'-azino-di-(3-ethyl-benzthiazoline-G-sulfonicacid) (ABTS).

Radioactive elements are also useful labeling agents and are usedillustratively herein.

An exemplary radiolabeling agent is a radioactive element that producesgamma ray emissions. Elements which themselves emit gamma rays, such as¹²⁴ I, ¹²⁵ I, ¹²⁸ I, ¹³¹ I, ¹³² I, and ⁵¹ Crrepresent one class of gammaray emission-producing radioactive element indicating groups.Particularly preferred is ¹²⁵ I. Another group of useful indicatinggroups are those elements such ¹¹ C, ¹⁸ F, ¹⁵ O and ¹³ N whichthemselves emit positrons. The positrons so emitted produce gamma raysupon encounters with electrons present in the animal's body. Also usefulis a beta emitter, such as ¹¹¹ indium.

The assay methods and systems of the present invention can thus utilizeor be comprised of a receptor of this invention affixed to solid matrixto form a solid support.

The antigen or receptor is typically affixed to the solid matrix byadsorption from an aqueous medium although several modes of adsorption,aswell as other modes of affixation, well known to those skilled in theart can be used. Exemplary of such modes are the reaction of thereceptor or antigen with the reactive carboxyl functionality produced bythe reaction of cyanogen bromide with glucose-containing matrices suchas cross-linked dextrose or cellulose, gluteraldehyde a linking asdiscussed hereinafter in conjunction with latex particles and the like.

Useful solid matrices are well known in the art. Such materials includethecross-linked dextran available under the trademark Sephadex fromPharmacia Fine Chemicals (Piscataway, NJ); agorse; beads of polystyrenebeads about 1 u to about 5 mm in diameter available from AbbottLaboratories of North Chicago, IL; polyvinyl chloride, polystyrene,cross-linked polyacrylamide,nitrocellulose or nylonbased webs such assheets, strips or paddles; or tubes, plates or the wells of a microtiterplate such as those made from polystyrene or polyvinylchloride.

Latex particles useful in agglutination-type assays are also usefulsolid matrices. Such materials are supplied by the Japan SyntheticRubber Company of Tokyo, Japan, and are described as carboxy-functionalparticlesdispersed in an anionic soap. Typical lots of such particleshave an average diameter of 0.308 microns (u), and have an averagecarboxy-functional group distribution of about 15 to about 30 squareAngstroms per carboxy group.

Prior to use, the particles are reacted with a diamine such as1,3-diamino-2-propanol to form a plurality of amide bonds with theparticle carboxy groups while maintaining free amine groups. The freeamines are thereafter reacted with a dialdehyde such as glutaraldehydeandthe receptor or antigen to form Schiff base reaction products. TheSchiff base reaction products are thereafter reduced with awater-soluble reductant such as sodium borohydride to provide a usefulsolid support.

Those skilled in the art will understand that there are numerous methodsofsolid phase immunoassays that may be utilized herein. Exemplary,useful solid phase assays include enzyme multiplied immunoassaytechniques (EMIT)and fluorescence immune assays (FIA), in addition tothe specifically discussed RIAs and ELISAs. However, any method thatresults in a detectable reaction of apoprotein B-100 with receptormolecules of this invention is considered part of this invention. Eachof those assay methods can employ single or double antibody techniquesin which an indicating means is utilized to signal the immunoreaction,and thereby thebinding of any apoprotein B-100 that is to be assayedwith a receptor of this invention. Exemplary techniques can be foundexplained in Maggio, Enzyme Immunoassay, CRC Press, Cleveland, OH(1981); and in Goldman, Fluorescent Antibody Methods, Academic Press,New York, NY (1980).

One embodiment of a specific method for assaying a body fluid sample forapo B-100 using a solid phase-affixed receptor of this invention is anoncompetitive ELISA wherein the immunoreactions are performedsequentially. In such an assay an aliquot of MB47 receptors, e.g.,generally about 1 to about 500 ug, is affixed to the inner walls of amicrotiter well to form a solid support.

Any apo B-100 present in the body sample provided is then immunoreactedwith the solid phase-affixed receptors. This is accomplished by admixingaknown amount, e.g., about 10 to about 200 microliters (ml) of sample(that can be prediluted) such as serum of plasma in the microtiter wellwith thesolid phase-affixed receptors to form a solid/liquid phaseadmixture. The admixture is maintained under biological assay conditionsfor a predetermined time period sufficient for any apo B-100 present inthe sample to immunologically bind to the receptor molecules and form asolid phase immunoreactant. The solid and liquid phases are thereafterseparated, and the solid phase is typically rinsed to help assureremoval of nonspecifically bound materials.

Apo B-100 present as solid phase immunoreactant (i.e., apo B-100 boundto solid phase-affixed MB47 receptors) is then immunoreacted withenzyme-labeled second receptor molecules. This is accomplished byadmixinga predetermined amount, e.g., about 0.1 to about 10 ug ofenzyme-labeled second receptor molecules in aqueous buffer solution,preferably horseradish peroxidase (HRPO)-labeled MB24 receptors, to forma second solid/liquid admixture. The second admixture is maintained asdescribed hereinabove, thus forming a solid phase immunoreactant"sandwich" consisting of MB47 receptor, apo B-100 and enzyme labeledMB24 receptors.

After separating the liquid phase from the solid phase as previouslydescribed, an aliquot of chromogenic substrate, such aso-phenylenediamine(OPD) for HRPO is admixed in the microtiter wellcontaining the solid phaseimmunoreactant to form a third solid/liquidadmixture. This admixture is maintained under biological assayconditions for a predetermined time period sufficient for any enzymepresent as immunoreactant to convert a proportional amount of substrateinto a colored product. The optical density of the resulting coloredsolution is then measured and compared toresults obtained usingsolutions containing known amounts of reagent apo B-100.

The ability of the noncompetitive sequential ELISA described above andin more detail in the Materials and Methods section to detect apo B-100in a body fluid sample was examined. In those studies, aliquots oflipoprotein depleted human plasma (LDP) to which were admixed knownamounts of reagentapo B-100 served as control body fluid samples.

The results of that study, shown in Table 1 below demonstrate that theabove-described method can accurately assay clinically relevant amountsofapo B-100 present in a body fluid sample.

                  TABLE 1                                                         ______________________________________                                        Noncompetitive Sequential ELISA Studies                                       of Apo B-100 In Han Plasma                                                    Sample.sup.1                                                                          Actual.sup.2                                                                           Ref..sup.3                                                                              MB47/MB24.sup.4                                                                        MB24/B18.sup.5                            ______________________________________                                        B Low   40         32 ± 44.3                                                                          42.6 ± 5.0                                                                          31.7                                      B Normal                                                                              80        71 ± 103                                                                             82.3 ± 11.8                                                                        54.7                                      B High  150      126 ± 180                                                                              149 ± 24.7                                                                        119                                       T Low   22.3     12.6 ± 17                                                                             16.2 ± 0.62                                                                        15.4; 9.0                                 T Normal                                                                              44.8     25.4 ± 49.2                                                                          36.1 ± 9.4                                                                          22.9; 19.7                                T HIgh  89.5     64.0 ± 101                                                                           77.4 ± 7.4                                                                          34.9; 36.3                                A                73         82.9 ± 11.7                                                                        66.4                                      AM               76        67.1 ± 8.1                                                                          46.8; 61.0                                B                86.5      69.8 ± 4.6                                                                          68.5; 90.5                                C                145        129 ± 3.8                                                                          147.173                                   D                109       81.0 ± 7.4                                                                          76.3; 98.2                                K                95         98.4 ± 16.5                                                                        77.5; 88.9                                R                60        64.2 ±  7.5                                                                         51.3; 52.9                                Pool             81.3      78.5 ± 6.8                                                                          54.7; 65.9                                C1               40.1      47.9 ± 6.7                                                                          47.0; 68.7                                C2               74.8       88.6 ± 13.2                                                                        81.3; 98.2                                C3               70.4      64.5 ± 4.8                                                                          64.5; 82.9                                C4               39.9      56.7 ± 6.6                                                                          48.5; 58.0                                ______________________________________                                         .sup.1 Clinically low, normal and high apoprotein B100-containing control     solutions were obtained from Johnson & Johnson Biotechnology Center, Inc.    La Jolla, CA (B) and Tago, Inc., Burlingame, CA (T). Plasma samples were       obtained from clinically normal individuals (one or two letter                designations and C1-C4), and the pool represents an admixture of 10 such      samples.                                                                      .sup.2 Actual apoprotein B100 concentration as determined by total protei    added to the sample. All concentrations shown in the table are in units of     mg/dl.                                                                        .sup.3 The concentration of apoprotein B100 as determined using the           reference assay (Ref.) described hereinafter. Values are given as a three     standard deviation range.                                                     .sup.4 Apoprotein B100 concentrations (mean ± 3 standard deviations)       obtained using solid phaseaffixed MB47 receptors and HRPOlabeled MB24         receptors in the noncompetitive sequential ELISA as described                 hereinbefore.                                                                 .sup.5 Apoprotein B100 concentrations (replicate values shown where           performed) obtained using solid phase affixed MB24 receptors and              HRPOlabeled B18 receptors (Curtiss et al., J. Biol. Chem., 257,               15213-15221 (1982)) in the noncompetitive ELISA wherein the                   immunoreactions were performed sequentially with the solid phase              immunoreactant being formed first.                                       

Another embodiment of a specific method for assaying a body fluid samplefor apo B-100 is a noncompetitive ELISA wherein the immunoreactions areperformed substantially simultaneously. To the before-describedmicrotiterwell containing solid phase affixed MB47 receptors is admixedsubstantiallysimultaneously the provided body sample and enzyme-labeledsecond receptorsthat immunoreact with a second apo B-100 epitope and donot substantially inhibit the binding of MB47 receptors to apo B-100.Such enzyme labeled second receptors are preferably MB24 receptors.

The resulting solid/liquid admixture is then maintained, separated andthe amount of enzyme present as part of the solid phase boundimmunoreactant sandwich determined as previously described.

The above described noncompetitive ELISA was used to examine the apoB-100 content of plasmas from 20 patients with coronary artery disease(CAD), 20patients with familial hypercholesterolemia and 20 normalsubjects. As shown in Table 2 below, the mean plasma apo B-100 level ofthe normal subjects was determined to be 85 milligrams/deciliter (mg/dl)with a 2 standard deviation range of ±21 mg/dl. This value is in closeagreementwith the normal range of apo B values reported by Curry et al.,Clin. Chem.24, 280-286 (1987), and Rosseneu et al., Clin. Chem. 28,427-433 (1983) each using different immunoassays.

Furthermore, the plasma apo B-100 levels in patients with CAD andhypercholesterolemia were higher than the upper limit of the 2 standarddeviation range found for normals. Similar results using othertechniques for CAD and hypercholesterolemia patients have been reportedby Sniderman et al., Proc. Natl. Acad. Sci. USA 77, 604-608 (1980) andLipid Research Council, JAMA 251, 351-364 (1984).

                                      TABLE 2                                     __________________________________________________________________________    Simultaneous ELISA Studies of Apo B-100 In Human Plasma                       Lipoprotein Levels.sup.2                                                                             Apoprotein B Levels.sup.3                                   Total.sup.3                                                                       HDL.sup.4                                                                          LDL.sup.5                                                                         Compet..sup.6                                                                      Non-Compet..sup.7                                                                     RID.sup.8                                                                          RID.sup.9                                 Subjects.sup.1                                                                     CHol.                                                                             Chol Chol.                                                                             ELISA                                                                              ELISA   #1   #2                                        __________________________________________________________________________    Normal                                                                             186 60   111 85   82      102  69                                        ±SD                                                                              37 16    30 21   22      20   19                                        CAD  205 37   133 109  104     138  84                                        ±SD                                                                              35  7    37 24   23      20   24                                        FH   331 37   270 196  204     211  136                                       ±SD                                                                             90  11    96 40   49      64   44                                        __________________________________________________________________________     .sup.1 Subjects include 20 normolipidemic, healthy controls (normal); 20      subjects with coronary artery disease defined by cardiac catheterization      (CAD); and 20 subjects with familial hypercholesterolemia (FH).               .sup.2 Values given are mean ±2 standard deviations from mean (±SD)     in units of mg/dl.                                                            .sup.3 Total cholesterol.                                                     .sup.4 Cholesterol present as HDL.                                            .sup.5 Cholesterol present as LDL.                                            .sup.6 Apo B100 level as determined by the competitive (Compet.) ELISA        described in the Materials and Methods section.                               .sup.7 Apo B100 level as determined by noncompetitive (NonCompet.) ELISA      using solid phase affixed MB47 receptors and HRPOlabeled MB24 receptors       wherein the sample and receptors were substantially simultaneously            admixed.                                                                      .sup.8 Apo B level determined using the radial immunodiffusion kit model      Diffugen RID available from Tago, Inc., Burlingame, CA                        .sup.9 Apo B level determined using the radial immunodiffusion kit model      MPartigen RIA available from CalbiochemBehring, La Jolla, CA             

The effect of diluting the body fluid sample prior to assaying it forapo B-100 by a noncompetitive simultaneous ELISA method was alsoexamined. Those results, shown in Table 3 below, indicate there was nosignificant difference in the apo B-100 levels determined over a 5 foldrange of plasma dilutions, i.e., 1:1000 to 1:5000.

                  TABLE 3                                                         ______________________________________                                        Simultaneous ELISA Studies of Apo B Levels                                    Determined from Different Dilutions of Plasma                                 Plasma Dilution                                                                         Plasma 1.sup.1                                                                             Plasma 2.sup.1                                                                          Plasma 3.sup.1                               ______________________________________                                        1:1000    54.4         136.6     199.0                                        1:2500    57.5         142.5     194.2                                        1:5000    53.0         126.0     178.0                                        Mean.sup.2                                                                              55.0         135.0     190.0                                        S.D..sup.3                                                                              1.88         6.83      8.98                                         % C.V..sup.4                                                                            3.4          5.0       4.7                                          ______________________________________                                         .sup.1 Plasma apo B100 concentration in units of mg/dl.                       .sup.2 Mean plasma apo B100 concentration for all 3 dilutions.                .sup.3 One standard deviation.                                                .sup.4 Percent coefficient of variance between results obtained for the       various dilutions.                                                       

Correlation between LDL cholesterol and plasma apo B-100 levels asdetermined by a noncompetitive ELISA for the normal and patient samplesdescribed above is shown in FIG. 7. The correlation coefficient of 0.89issimilar to that reported by Albers et al., Metabolism 24, 1339-1351(1975) and Slater et al., Clin. Chem. 31, 841-845 (1985).

Embodiments of the assay methods of this invention using a solid supportcontaining reagent apo B-100 affixed to a solid matrix affixed to asolid matrix are performed using the following steps:

(a) A body fluid sample containing apo B-100 is provided as describedhereinbefore.

(b) Substantially simultaneously admixing

(1) the fluid sample;

(2) a predetermined amount of either MB24 or MB47 receptor molecules;and

(3) a predetermined amount of solid phase-affixed reagent apo B-100, toform a liquid/solid phase immunoreaction admixture.

The admixture is maintained under biological assay conditions for a timeperiod sufficient for the antibody combining sites of the receptormolecules to immunoreact with (immunologically bind to) either thereagentapo B-100 or any apo B-100 present in the body fluid sample. Thereceptor molecules immunologically binding apo B-100 present in the bodysample form a liquid phase immunoreactant and those binding solidphase-affixed reagent apo B-100 form a solid phase immunoreactant.

(c) The presence of the solid phase-affixed immunoreactant is thenassayed and the amount of apo B-100 present in the sample is therebydetermined bycomparison of the amount of binding exhibited by a knownamount of MB47 or MB24 admixed with similar solid phase-affixed apoB-100, as discussed hereinafter. Preferably, the immunoreactant assay isperformed after the liquid phase and solid phase immunoreactant of step(b) are separated, as by washing. The amount of sample apo B-100 boundas liquid phase immunoreactant can be determined as by the use oflabeled antibodies that immunoreact with the receptor molecules of thisinvention such as peroxidase-linked goat anti-mouse Ig, or as otherwisedescribed herein.

In a particularly preferred embodiment of the competition assay, about 1ugto about 10 ug of reagent apo B-100 are affixed to a solid matrix,preferably the walls of a microtiter well, to form a solid support. Anynonspecific binding sites on the solid support are typically blockedwith a protein such as BSA or the like.

A predetermined amount of receptor molecules of this invention, e.g.,generally about 0.1 ug to about 10 ug, is substantially simultaneouslyimmunoreacted with the solid phase-affixed reagent apo B-100 and any apoB--100 present in a body fluid sample (which can be diluted) such asserumor plasma. This is accomplished by substantially simultaneouslyadmixing analiquot of sample and the receptor molecules in themicrotiter well containing solid phase-affixed reagent apo B-100.

The solid/liquid admixture thus formed is maintained under biologicalassayconditions for a time period sufficient for the receptor moleculespresent to immunologically bind the apo B-100 present. The solid andliquid phasesare preferably thereafter separated as by washing and thesolid phase is typically rinsed to help assure removal ofnon-specifically bound materials.

The presence of solid phase-affixed immunoreactants is then assayedtypically by use of labeled antibodies that immunologically bind thereceptor molecules of this invention such as peroxidase-labeled goatanti-mouse IgG.

In the competitive ELISA, apo B-100 present in the patient samplecompetes with a constant, known amount of reagent apo B-100 for aconstant, known number of receptor molecule antibody combining sites inthe immunoreactionadmixture. Competition provided by the sample apoB-100 results in a decrease of detectable solid phase-affixedimmunoreactant; the greater thedecrease, the greater the amount of apoB-100 present in the body sample under investigation.

To obtain relative amounts of apo B-100 present in patient plasmas,resultsobtained using apo B-100 present in patient plasma as competitorswere compared to results obtained using competitive standards containingknown amounts of reagent apo B-100. A standard curve was prepared usingLDL concentrations ranging from 32 mg/ml to 0.25 mg/ml (320 mg/dl to 2.5mg/dl).

The competitive ELISA described above was used to examine the samenormal and patient samples evaluated by noncompetitive ELISA. Thoseresults, alsoshown in Table 2 hereinbefore, are in close agreement withthe results obtained by the noncompetitive assay.

The effect of diluting the body fluid sample prior to assaying it forapo Bin the above-described competitive ELISA was examined. Thoseresults, shownin Table 4 below, demonstrate no significant difference inthe apo B levelsdetermined over a 4-fold range of plasma dilutions;i.e., 1:100 to 1:400.

                  TABLE 4                                                         ______________________________________                                        Competitive ELISA Studies of Apo B Levels                                     Determined From Different Plasma Dilutions                                    Plasma Dilution                                                                           Plasma 1.sup.1                                                                            Plasma 2 Plasma 3                                     ______________________________________                                        1:100       32.8        63.3     175.8                                        1:200       30.0        58.5     183.2                                        1:300       31.8        64.8     190.9                                        1:400       33.8        62.6     190.0                                        Mean.sup.2  32.1        62.3     185.0                                        S.D..sup.3  1.6         2.7       7.0                                         % C.V..sup.4                                                                              5.0         4.3       3.8                                         ______________________________________                                         .sup.1 Plasma apo B100 concentration in units of mg/dl.                       .sup.2 Mean plasma apo B100 concentration for all 3 dilutions.                .sup.3 One standard deviation                                                 .sup.4 Percent coefficient of variance between results obtained for the       various dilutions.                                                       

Correlation between LDL cholesterol and plasma apo B levels asdetermined by the competitive ELISA for the normal and patient samplesdescribed above is shown in FIG. 8. The correlation coefficient of 0.92is similar to that reported by Albers et al., Metabolism, 24, 1339-1351(1975), and Slater et al., Clin. Chem., 31, 841-845 (1985), thusindicating that the competitive ELISA accurately predicts circulatingLDL cholesterol levels.

The correlation between apo B-100 levels obtained using the competitiveandnoncompetitive ELISAs was examined. As shown in FIG. 9 there was ahigh correlation (r=0.92) between the results obtained in each assay.

A diagnostic system, preferably in kit form, useful for carrying out theabove assay methods includes, in separate packages, (a) a first specificbinding agent wherein said agent is (i) a receptor that immunoreactswith apo B-100, and is selected from the receptor secreted by hybridomaHB 8746(MB47) or the receptor secreted by hybridoma HB 8742, (MB24) and(b) a labeled second specific binding agent for signaling theimmunoreaction of said first binding agent with apo B-100. Preferably,the labeled specific binding agent is a receptor linked to an enzyme.More preferably, the labeled agent is the remaining of either receptorof (a) above, linked to an enzyme.

In preferred embodiments, the system further includes another containerof reagent apo B-100 for use as control and/or target antigen. Alsopreferredare embodiments wherein the system includes a solid matrix towhich the first specific binding agent or reagent apo B-100 is affixedto form a solid support. Useful solid matrices are as already described.Preferably,however, the solid matrix is the well of a microtiter plate.

Known amounts of the specific binding agents are provided. Those amountsare at least enough to carry out one assay. The provided specificbinding agents are typically supplied in a form and amount that isdesigned to be diluted to a prescribed volume with water, saline or abuffer such as phosphate-buffered saline at pH 7.3-7.5.

Additional packages can also be included in the system. Such packagescan contain (i) buffer salts in dry or liquid form, (ii) enzymesubstrates such as o-phenylenediamine, and the like.

Exemplary packages include glass and plastic such as polyethylene andpolypropylene bottles or vials; plastic, plastic-metal foil,plastic-metalfoil-paper envelopes and the like. The specific bindingagents can be packaged in an aqueous liquid form as in ascites orbuffer, but preferably, they are supplied in dried form such as thatprovided by lyophilization.

D. Affinity Sorbants

The present invention also contemplates an affinity sorbant, preferablysterile, comprised of a biologically active receptor of this inventionaffixed to a solid matrix to form a solid support. The solid matrix ispreferably in particulate form. Such affinity sorbents are useful forspecific removal of apoprotein B-100 containing lipoprotein (VLDL andLDL)by immunoadsorption from plasma of patients suffering fromhypercholesterolemia.

The solid support can be a wide variety of materials such ascross-linked dextran, e.g., Sephadex G-25, -50, -100, -200 and the likeavailable from Pharmacia Fine Chemicals of Piscataway, N.J., agarose andcross-linked agarose, e.g., Sepharose 6B, CL6B, 4B, CL4B and the likealso available from Pharmacia Fine Chemicals or Bio-Gel A-0.5M, A-1.5M,A-50M and the like available from Bio-Rad Laboratories, Richmond CA, orpolyacrylamide beads, e.g., Bio-Gel P-2, P-30, P-100, P-300 and the likealso available from Bio-Rad Laboratories. The agarose and cross-linkedagarose materials are preferred herein because of their high porosityand low non-specific binding properties and will be used illustrativelyas a solid matrix.

Sterilization of the receptors and solid matrix is typically performedbefore linking. To maintain biological activity, receptors are usuallysterilized by filtration, for example by passage through a 0.22 micronnitrocellulose filter. Sterilization of the matrix depends, as is wellknown, on the type of matrix. For example, Sepharose cannot besterilized by autoclaving, but it can be sterilized chemically, forexample, by treatment with diethylpyrocarbonate. On the other hand,cross-linked Sepharose can be sterilized by autoclaving at pH 7, 120degrees C. for 20 minutes.

The Sephadex or Sepharose matrix is typically activated for linkingusing cyanogen bromide by methods well known in the art. The activatedmatrix isthen washed and linked to receptors that immunoreact with apoB-100 and aresecreted by hybridoma HB 8746 or hybridoma HB 8742. Thematrix-linked receptor is then washed and is ready for use. Unreactedreactive groups onthe support can be reacted with an amine such asethanolamine or Tris if desired.

The affinity sorbant can be used in its loose state but is preferablyconfined in a column. Plasma from a patient containing apo B-100 is thenadmixed with the affinity sorbant to form an immunoreaction admixture.Theadmixture is maintained under biological assay conditions for a timeperiodsufficient for the solid matrix-affixed receptors toimmunologically bind apo B-100 present in the plasma and form a solidmatrix-affixed immunoreactant. The plasma is then separated from thesolid matrix-affixedimmunoreactant. The apo B-100 (LDL andVLDL)-depleted plasma so produced can then be introduced into thepatient from which it was originally obtained.

The preparation of an affinity sorbant and its use for removal ofapolipoprotein B contained lipoproteins from plasma are described inStoffel et al., Proc. Natl. Acad. Sci. USA 78, 611-615 (1981), which isincorporated herein by reference. An affinity adsorbant column preparedbylinking substantially pure MB47 receptors to CNB4-activated Sepharose4B was found to immunoadsorb (bind) 3mg of LDL per ml of solid support.

II. Materials Methods A. Preparation of Human and Lipoproteins

Human lipoprotein fractions were isolated by centrifugation at thefollowing densities: VLDL, density (d) less than 1.006 gm/ml; LDL, dequalto 1.025-1.050 gm/ml; HDL, d equal to 1.070-1.21 gm/ml, and LDS, dequal to1.21 gm/l). In some cases, human LDL was also isolated at d1.019-1.063 gm/ml or at 1.045-1.065 gm/ml. The protein concentration ofplasma and each fraction were determined by the Lowry technique [Lowryet al., J. Biol. Chem. 193, 265-275, (1951)] as modified by Markwell etal., Anal. Biochem. 87, 206-210 (1978), using a BSA standard.

For each LDL and VLDL fraction, estimations of apo B content were alsomadefollowing precipitation of apo B with tetramethylurea (TMU) usingthe technique of Kane et al., J. Clin. Invest., 56, 1622-1634, (1975))or isopropyl alcohol following the teachings of Equsa et al., J. LipidRes., 24, 1261-1267 (1983).

Fresh, fasting human plasma was obtained from normal healthy donors byplasmaphoresis, and was adjusted to 0.1 percent EDTA (s/v). Pools madeup from three or more donors were used unless otherwise stated. Thelipoproteins were isolated by sequential ultracentrifugation of theplasmausing solid KBr for density (d) adjustment. The lipoproteinfractions included VLDL, d less than 1.006 g/ml; IDL, d = 1.006-1.019g/ml; LDL, d =1.019-1.063 g/ml; and HDL, d = 1.063-1.25 g/ml.

The bottom fraction containing lipoprotein-depleted serum (d greaterthan 1.25 g/ml) was also collected.

The fractions were dialyzed thoroughly against lipoprotein buffercontaining 0.15 M NaCl, 0.3 mM EDTA, 0.0005 percent alpha-tocopherol ata pH value of 7.4.

A chylomicron fraction was separated from the VLDL fraction ofnon-fasting pooled plasma by floating the chylomicrons throughlipoprotein buffer withultracentrifugation at 120,000×g for 40 minutesat 4 degrees C.

All lipoproteins were filter-sterilized, and stored at 4 degrees for nomore than 20 days. Lipoproteins were analyzed for protein content by amodification of the method of Lowry, J. Biol. Chem. 193, 265-275 (1951),using a BSA standard. All lipoprotein concentrations are expressed onthe basis of protein.

The apoprotein composition of each of the lipoprotein classes wasassessed by SDS-polyacrylamide gel electrophoresis. The chylomicronscontained detectable amounts of apoproteins, B, E and C, while the VLDLcontained apoproteins B, E, C and trace amounts of apoprotein A-I.

The IDL contained apoproteins, B, E and C, while the LDL contained onlyapoprotein B. The HDL contained apoproteins AI, AII and C.

During the course of these studies, lipoprotein preparations displayed aconsistent apoprotein composition. To control for potential proteolysisofapoproteins, selected plasma pools were isolated and stored in thepresenceof 1 mg/ml gentamycin sulfate, 0.2 percent sodium azide, and 1mM benzamidine, 10 mM diisopropyl fluorophosphate, 10 ug/ml soybeantrypsin inhibitor.

Comparison of SDS-PAGE apoprotein-staining patterns of theselipoproteins, as well as those isolated in the absence of antibioticsand protease inhibitors immediately after ultracentrifugation and afterstorage for up to 3 weeks showed no evidence of proteolysis. Allpreparations that were used were sterile.

B. Hybridoma Production and Culture

Intact native lipoproteins isolated from pooled plasma were used forimmunization. Balb/c mice four to five weeks of age were immunizedintraperitoneally with 50 micrograms of lipoprotein in complete Freund'sadjuvant. Secondary intraveneous injections of 50 ug of lipoprotein inlipoprotein buffer were given between day 28 and 33. Spleens wereremoved from the immunized mice 72 hours after the last injection andsingle cell suspensions were prepared in HT medium. Blood was alsocollected, and the serum was used as a positive control for each of theimmunoassays.

The murine myeloma cell lines were maintained in log phase growth instationary cultures in complete HT medium [Kennet et al. Curr. Top.Microbiol. Immunol. 81, 77-91 (1978), which is incorporated herein byreference] containing 0.1 mM azaguanine. Fusions Were performed in thepresence of 30 percent (v/v) polyethylene glycol 1000 (Sigma, St. Louis,MO) at a ratio of immune spleen cells to P3×63Ag8 of 10:1. Three daysafter fusion, the cells were plated out in 96-well tissue culture platesat 1×10⁵ viable cells/well in HT medium containing 0.1 mM aminopterin.

The cells were fed 7 days after fusion with HT medium and atapproximately 4-5 day intervals thereafter as needed. Growth wasfollowed microscopically and culture supernatants were collected on day14 for assay of antigen-specific antibody production by a solid phaseRIA. Specific antibody-producing hybridomas were cloned 19 to 47 daysafter fusion by limiting dilution in the presence of Balb/c splenicfeeder cells, and hybridomas in wells containing single colonies werescreened for antibody production by solid phase RIA after 10 days. Thecloned hybridomas were cultivated in medium containing 10 percent calfserum, andwere stored frozen in liquid nitrogen.

C. Inhibition of LDL Binding, Internalization and Degradation

The ability of anti-apo B-100 receptors to inhibit binding,internalizationand degradation of ¹²⁵ I-LDL by human fibroblasts wasassessed in the following manner. LDL was iodinated with ¹²⁵ I (specificactivity 200cpm/ng) using the iodine monochloride technique of Bilheimeret al., J. Clin. Invest., 56, 1420-1430 (1975).

To allow antibody-LDL interaction, 0.1 ml of each hybridoma supernatantwasincubated (admixed and maintained) with ¹²⁵ I-LDL (finalconcentration2.5 mg/ml) in 0.4 ml of Dulbecco's minimal essential medium(DME) containing 2.5 mg/ml lipoprotein-deficient serum (LDS) for 12hours at 4 degrees C. Then, the individually incubated mixtures weretransferred to human foreskin fibroblast monolayers grown in DME with 10percent fetal calf serum (FCS) in 10 millimeter-diameter wells. LDLreceptors of these cells had been maximally expressed by priorincubation of those cells for a time period of 24 hours in DMEcontaining 2.5 mg/ml LDS.

After individual incubations of supernatant and fibroblast cells for 6hours at 37 degrees C., the cellular degradation of ¹²⁵ I-LDL wasassessed in accordance with the method of Drevon et al., J. Lipid Res.,22, 37-46 (1981). Control cultures had 0.4 ml of DME containing ¹²⁵I-LDL and one of the following: a) 0.1 ml of fresh hybridoma mediumcontaining 20 percent fetal calf serum; or b) 0.1 ml hybridoma mediumfromwells of hybridoma colonies that were negative for apo B-specificantibody production; c) 0.1 ml DME containing 2.5 mg/ml LDS; or d) 0.1ml unlabeledLDL (to bring final unlabeled LDL concentration in the mediato 500 ug per ml).

D. Isolation of Anti-LDL Immunoglobin

Ascites fluids containing receptors of this invention were obtained from10-week-old Balb/c mice, which had been primed with 0.3 ml of mineraloil and injected intraperintoneally with 3-50×10⁵ hybridoma cells. Theaverage time for development of ascites was 12 days. Followingclarification by centrifugation at 15,000×g for 1 hour at 4 degrees C.,ascites fluids were pooled and stored frozen at -20 degrees C.

Isolated antibody MB47 was prepared by chromatography of the monoclonalhybridoma ascites fluids on a protein A-Sepharose 4B column (PharmaciaFine Chemicals, Piscataway, New Jersey). Antibody was eluted from thecolumn with 0.1 molar (M) acetic acid.

Isolated receptors also were prepared by fast protein liquidchromatography(FPLC) of a monoclonal hybridoma ascites fluid on aPharmacia Mono Q HR 5/5anion exchange column in a Pharmacia FPLC Systemusing a 0-0.5M NaCl gradient in 10mM Tris, pH 8.0, and following thedirections supplied with the column.

E. Characterization of Hybridoma Antibodies

The total gamma-globulin (Ig) content of each pool of ascites fluid wasobtained by electrophoresis of 1-3 ml samples of cellulose acetatestrips in 75 mM veronal buffer, at a pH value of 8.6 for 45 minutes at200 millivolts (mV). The percentage of the total protein that was Ig wasquantitated by densitometric scanning of the Ponceau S-stained gels, andtotal protein was determined by the modified Lowry methods as discussedbefore.

Murine Ig heavy and light chains were identified by double diffusion in0.9percent agarose. Ten microliters of an appropriate dilution ofascites fluid were reacted with an equal volume of appropriately dilutedrabbit anti-murine heavy and light chain-specific antisera (LittonBionetics). Following diffusion for about 15 hours at 20 degrees C. andwashing, precipitin lines were identified by staining with 0.5 percentCoomassie brilliant blue R-250.

Isoelectric profiles of each monoclonal antibody were obtained byisoelectric focusing of 0.01 ml samples of the ascites fluids in 0.8percent agarose (EF 802-300 LKB) containing 10 percent sorbitol, 2percentampholine within a pH value range of 5-8, (LKB) for 150 minutesat 3 watts constant power. Following fixing and drying, the gels werestained with Coomassie brilliant blue and photographed.

1. Western Blotting -- Apoproteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of thelipoproteins in a vertical slab gel apparatus (14×12×0.15 cm) (HoefferScientific Instruments, San Francisco, CA). The gels were prepared usinga25 mM Tris-glycine buffer at a pH value of 8.6. The upper stacking gelcontained 1 percent SDS, 3 percent acrylamide, and the lower running gelwas either a 3 to 20 percent or a 3 to 6 percent acrylamide gradientcontaining 1 percent SDS. The lipoproteins were delipidated by boilingfor3 minutes in electrophoresis sample buffer that contained 1 percentsodium dodecyl sulfate, 10 mM Tris, and 0.24 mM EDTA. Molecular weightmarkers and their respective apparent relative molecular masses were:fibrinogen, 340,000; IgG, 140,000; albumin, 69,000; ovalbumin, 43,000;soybean trypsininhibitor, 20,500; and lysozyme, 14,300. The gels wereelectrophoresed for approximately 18 hours at 13.5 milliamps (mA)constant current.

The gels were washed in distilled water for 10 minutes and then for 10minutes in 25 mM Tris, 192 mM glycine, pH 8.3, containing 20 percent(v/v)methanol. Transfer to nitrocellulose (0.45 micro, Millipore Corp.)was accomplished by electrophoresis for 1 hour at 400 mA. Remainingactive binding sites on the nitrocellulose were saturated by soakingovernight inPBS containing 3 percent BSA, 3 percent normal goat serum,0.01 percent sodium azide (blocking solution).

The fixed gels or the nitrocellulose transfers were incubated for 18hours at 4 degrees C. with either immune mouse serum or ascites fluidappropriately diluted in PBS containing 3 percent BSA, 3 percent normalgoat serum, 0.05 percent Tween-20 (polyoxyethylene (20 monolaurate).Afterrepeated washing, antibody binding was detected by a second 4 hourincubation at 4 degrees C. with ¹²⁵ I-goat anti-immune Ig (0.5microCi/ml) in the same buffer followed again by extensive washing.

Nonspecific binding to the nitrocellulose was significantly reduced bywashing after incubation with both the first and second antibodies inPBS containing 3 percent BSA, 0.05 percent Tween-20 and then in 0.5 MLiCl containing 0.1 percent SDS.

The gels or nitrocellulose transfers were dried and analyzed byautoradiography (X-Omat; Eastman Kodak) at -20 degrees C. Whereappropriate, gels were stained with either 0.1 percent Coomassiebrilliantblue R-250 in 50 percent trichloroacetic acid or silver stain(Bio-Rad) as described by Merril et al., Proc. Natl. Acad. Sci. USA, 76,4335-4339 (1979).

F. Preparation of Fab Fragments

Fab fragments of isolated receptor molecules antibody were formed bydigestion with papain. The antibody Fc portions and undigestedantibodies were removed by passage over a protein A-Sepharose 4B column.SDS-PAGE of the Fab fragments revealed two discrete bands of 25,000 and40,000 daltons. Immunoreactivity of the Fab fragments was verified byspecific binding to LDL in a solid phase RIA (Milne et al.,Arteriosclerosis 3, 23-30 (1983)).

G. Radioimmunoassays (RIA) 1. Fluid Phase ¹²⁵ I-Labeled Antigen RIA

To determine the fraction of ¹²⁵ I-LDL particles bound by MB47 and MB24,a fluid phase RIA was utilized [Curtiss and Edgington, J. Biol. Chem.,25, 15213-15221 (1982)]. Two different LDL (d=1.019-1.063 gm/ml)preparations were studied, one isolated from pooled plasma of 10 normalsubjects and one isolated from plasma of one normal subject. ¹²⁵ I-LDL(2000 cpm/ng), prepared using the Iodogen (Pierce Chemical Co.,Rockford, IL) technique, was 90 percent trichloracetic acid (TCA)precipitable. It was diluted in 9 percent bovine serum albumin (BSA)(Sigma, St. Louis, MO) and centrifuged at 30,000×g for 15 minutes priorto each assay to remove complex material. Assays were performed in 12×75nun glass tubes in triplicate in 55 mM sodium barbital buffer,at a pHvalue of 8, containing 150 mM NaCl, 0.02 percent sodium azide, 3 percentBSA, and 1.5 mM sodium-EDTA. To 0.1 ml of ¹²⁵ I-LDL (containing 20 ngLDL protein) were added 0.1 ml of buffer or competing antigen and 0.1 mlof increasing concentrations of isolated MB47 receptorsdiluted in theBSA-barbital buffer. After 18 hours at 4 degrees, 0.1 ml of IgSorb (TheEnzyme Co., Boston, MA) were admixed. After 2-hours of maintenance time,2 ml of BSA-free barbital buffer were added, and the tubes wereimmediately centrifuged at 1,500×g for 60 minutes. The precipitates werewashed twice with barbital buffer. Maximum precipitable radioactivitywas determined by replacing the IgSORB with 100 percent TCA.The minimumprecipitable radioactivity was determined in the absence of MB47receptors. The percent ¹²⁵ I-LDL bound was then calculated.

2. Fluid Phase ¹²⁵ I-labeled Receptor RIA

Intact antibody MB47 receptors, isolated by immunopurification, wereiodinated with ¹²⁵ I using the Iodogen technique (specific activity 3000cpm/ng) (Pierce). Following extensive dialysis against PBS, over 95percent of the radioactivity was precipitable by 10 percent TCA. Greaterthan 98 percent of the ¹²⁵ I-MB47 bound to a human LDL affinity column.Assays were performed in triplicate in 10×75 mm silicone-coated glasstubes. Increasing concentrations of ¹²⁵ I-MB47 in 0.1 ml of BSA-barbitalbuffer were added to 100 ng of pooled, normal human LDL diluted in 0.2ml of BSA-barbital buffer. Each tube contained 182 fmoles of LDL apo B(assuming an apo B molecular weight of 550,000 daltons). Aftermaintenance for 16 hours at 4 degrees C., LDL was quantitativelyprecipitated by a lipoprotein depleted rabbit antiserum specific forhuman LDL. (Only the density greater than 1.21 gm/ml fractionof therabbit antiserum was used because antibody MB47 binds rabbit apo B.)

Preliminary studies established a concentration of delipidated rabbitantiserum that precipitated 97 percent of 100 ng of ¹²⁵ I-human LDL.After admixture of the rabbit antibody the tubes were maintained for 16hours at 4 degrees C., and then spun at 1500×g for 50 minutes at 4degrees C. The supernatants were removed, and the pellets were washedtwice with 2 ml of ice cold barbital buffer. Nonspecific binding andprecipitation were determined in two sets of parallel tubes.

In the first set, no human LDL was added to the initial incubation, butthesame amount of rabbit second antibody was added. In the second set oftubes, non-immune rabbit serum (density greater than or equal to 1.21gm/ml fraction) was substituted for the immune rabbit serum antibody.

Both methods yielded identical values for nonspecific binding, which waslinear with increasing concentrations of ¹²⁵ I-MB47 antibody, and in allcases was less than 1 percent of the total counts added. Specific ¹²⁵I-MB47 binding to LDL was obtained by subtracting nonspecific bindingfrom total binding. Binding data were analyzed utilizing a linearregression program for Scatchard analysis of ligand binding systems,that provided an estimate of the antibody affinity constant (Ka) and thereceptor or epitope concentration (Munson et al., Anal. Biochem., 107,220-239 (1980)).

3. Solid Phase-Affixed Antigen RIA

A screening assay to determine whether a hybridoma culture was producingreceptors of this invention was performed in flexible round bottompolyvinyl chloride microtiter plates (Dynatech, Inc., Alexandria, VA) assolid matrix. Lipoprotein antigens (reagent apo B-100) were affixed(bound) to the inner wells of the microtiter plate wells by admixing0.05 ml of lipoprotein in lipoprotein buffer, and incubating the platesat roomtemperature for 3 hours to provide a constant final bound antigenconcentration, and the solid support. Preliminary direct binding studiesusing radioiodinated lipoproteins indicated that there were significantdifferences in the efficiencies with which each of the lipoproteins wasbound to the plastic microtiter wells. Therefore, to achieve a finalboundantigen concentration of 50 ng of protein/well, VLDL was used at 50ug/ml, IDL at 6.2 ug/ml, LDL at 4.4 ug/ml, and HDL at 24 ug/ml.Nonspecific binding sites were then blocked by admixing of 0.25 ml ofblocking solution in each well, maintaining the admixture for one hour,and then separating the blocking solution from the wells thereby forminga solid support with low nonspecific binding capacity.

For assay, 0.050 ml of antiserum, culture supernatant, or ascites fluiddiluted in PBS containing 3 percent BSA, 3 percent normal goat serum,and 0.05 percent Tween-20 [polyoxyethylene (20) sorbitan monolaurate]were admixed in the wells to form solid/liquid phase immunoreactionadmixtures.The admixtures were then maintained (immunoreacted) for 18hours at 4 degrees C.

After washing the wells with PBS containing 3 percent BSA and 0.05percent Tween-20 to remove nonbound material solid phase-bound receptorswere prepared for direct detection by admixing 10 ng of immunochemicallypurified and radioiodinated goat anti-murine Ig in each well to form asecond solid/liquid phase admixture. This admixture was maintained for 4hours at 4 degrees C. to allow the labeled second receptors to bind thesolid phase-bound first receptors and form a sandwich immunoreactant.

After a final wash to remove nonbound labeled receptors, individualwells were removed and counted for ¹²⁵ I, the amount of ¹²⁵ Idetectedbeing in direct proportion to the amount of first receptorsbound as solid phase immunoreactant.

The immunochemically purified second antibody was radioiodinatedenzymatically using immobilized lactoperoxidase and glucose oxidase(Enzymobeads, Bio-Rad Burlingame, CA) to specific activities of 3-4microCi/microgram.

4. Competitive Solid Phase Affixed Receptor RIA

Competitive solid phase radioimmunoassays (RIAs) for human apo B wereperformed using antibody MB47. Polyvinyl chloride wells were coated with0.5 ml of human LDL (reagent apo B-100) diluted to 10 ug/ml in PBS, pH7.35, and maintained for 2 hours at 37 degrees C. Nonspecific bindingsites were blocked by coating with 5 percent BSA in PBS for 30 minutesat room temperature. Plates were then washed with PBS washing bufferadditionally containing 0.1 percent BSA, 0.01 percent sodium azide and0.05 percent Tween-20.

Fresh human LDL, prepared from pooled normal plasma, was used as reagentapo B-100 for the standard curve in dilutions ranging from 0.4 ug/ml to97.2 ug/ml. All dilutions were made in a PBS buffer containing 3 percentBSA, 0.01 percent sodium azide, and 0.05 percent Tween-20. The standardLDL or competitors (0.025 ml) were admixed to the LDL coated wellsfollowed by 0.025 ml of buffer containing a fixed and limiting amount ofmonoclonal antibody (ascites fluid). The optimal final concentration ofantibody was determined from preliminary antibody dilution studies, andwas chosen as the amount of antibody resulting in 50 percent of maximumbinding.

The plates were maintained for a time period of about 18 hours at 4degreesC., then washed with the PBS washing buffer. Mouse antibodybinding was then quantitated by admixture of 0.05 ml of ¹²⁵I-immunopurified goatanti-mouse Ig (450 ng/well, 8,000 cpm/ng). After a4 hour maintenance time period at 4 degrees C., the plates were washedand individual wells counted.

H. Enzyme-Linked Immunosorbant Assay (ELISA) 1. Noncompetitive ELISA

Isolated MB47 receptors were affixed to the walls of polystyrenemicrotiterplate wells (Nunc-Immuno Plate I) by admixing 0.15 ml of asodium bicarbonate buffer pH 9.0, containing 1 ug/ml receptor proteininto each well. The wells were maintained for 16 hours at 4 degrees C.,and then washed 3 times with PBS containing 0.1 percent BSA and 0.05percent Tween.Residual nonspecific binding sites were then blocked byadmixing 0.2 ml of 3 percent BSA in PBS in each well, maintaining theadmixture for 1 hour at23 degrees C. and then washing as describedabove. Wells (solid supports) so prepared can be used for up to aboutone month after preparation, when stored in a humidified chamber.

Reagent apo B-100 (human LDL) was diluted in PBS to concentrationsranging from 2.0 to 0,062 ug/ml for use as standard control solutions.Plasma samples were diluted 1:2000 in PBS.

Fifty microliters of standard or sample were admixed in the wells intriplicate. Within about 5 minutes thereafter, 50 ul of PBS containingmg/ml of HRPO-labeled MB24 receptors were admixed in each well. Theimmunoreaction admixtures were maintained 30 minutes at 25 degrees C.Nonbound material was then separated from the wells by washing asdescribed before.

The amount of solid phase affixed sandwich immunoreactant containingHRPO label was then assayed by admixing 0.1 ml of freshly preparedsubstrate solution (distilled water containing 3 percent H₂ O₂ and 0.67mg/ml o-phenylenediamine (OPD)) to each well. Color was allowed todevelopfor 30 minutes at 25 degrees C. The substrate conversion reactionwas then stopped by admixing into each well 0.05 ml of 4N H₂ O₂. Theoptical density (O.D.) of the solutions was determined at a 490nanometers(nm) wavelength using a Dynatech MR600 (Dynatech, Alexandria,VA) microtiter plate reader.

2. Competitive ELISA

Reagent apo B-100 was affixed to the walls of flexible polyvinylchloride microtiter plate wells (Microtest III, Falcon Labware, Becton,Dickinson &Co., Oxnard, CA) as solid matrix by admixing 0.2 ml of PBScontaining 5 ug/ml of isolated human LDL into each well. The wells weremaintained for 16 hours at 4 degrees C., and were then washed 3 timeswith 0.2 ml of PBS containing 1 percent BSA, 0.5 percent Tween and 0.02percent aprotinin (Sigma Chemical Co.). Residual nonspecific bindingsites were blocked as described in the noncompetitive ELISA.

For the standard curve, which was included on each plate, the reagentapo B-100 was diluted in PBS containing 0.5 percent lipoprotein-depletedplasma (LPDP) to provide concentrations ranging from 32 mg/ml to 0.25mg/ml.

Plasma samples were diluted 1:200 in PBS containing 0.5 percent LPDP.Fiftymicroliters of the standards or samples were admixed in triplicateinto thewells. Within about 5 minutes thereafter, 50 ul of PBScontaining 3 percentBSA and about 4 ug/ml of MB24 receptors were admixedinto each well. The admixtures so formed were maintained for about 18hours at 4 degrees C. The nonbound material was then separated from thesolid phase affixed MB24-reagent apo B-100 immunoreaction products bywashing as described above.

The solid phase immunoreactants were prepared for assaying by admixing0.1 ml of PBS containing 1 percent BSA and an effective amount ofHRPO-labeledgoat anti-mouse IgG to each well. This second immunoreactionadmixture was maintained for about 1 hour at 24 degrees C. and thenwashed as described above to form a sandwich immunoreactant.

The amount of solid phase affixed sandwich immunoreactant containingHRPO label was assayed as described in the competitive ELISA.

I. Plasma Samples and Lipoprotein Quantification

Plasma samples were obtained from 20 patients with coronary arterydisease from the cardiac catheterization laboratory at the San Diego VAHospital and from 20 patients with familial hypercholesterolemia from aUniversity of California, San Diego clinic. In addition, plasma wasobtained from 20 normal subjects.

Blood was collected into tubes containing 1.5 mg/ml ethylenediaminetetraacetate (EDTA), and the plasma was separated immediately bycentrifugation at 4 degrees C.

Total plasma cholesterol and triglycerides were measured on fresh plasmasamples in a standardized clinical laboratory using an Abbott ABA-200bichromatic analyzer, and Boehringer-Mannheim high performancecholesterolreagent 236691 and Abbott Laboratories triglycerides A-gent.LDL- and HDL-cholesterol were measured using techniques described inLipid ResearchClinic Procedures, HEW Pub. No. 75-628 (NIH), 2 ed.,Washington, D.C., Gov.Print. Off., (1974). Apoprotein B levels weredetermined using two commercially available radial immunodiffusion kits:Diffu-gen RID (Tago, Inc., Burlingame, CA) which is termed RID #1 here,and M-Partigen RID, (Calbiochem-Behring, La Jolla, CA) which is termedRID #2 herein.

J. LDL Binding to a MB47-Sepharose 4B Column

50 Milligrams of substantially pure MB47 receptors, obtained by proteinA column chromatography, were linked to 12 ml of cyanogenbromide-activated Sepharose 4B (Pharmacia Fine Chemicals, Piscataway,N.Y.), according to manufacturer's instructions. Subsequently, 10 mg ofLDL was added to the column and maintained overnight (about 16 hours) at4 degrees C. The nonbound LDL was then eluted and assayed for LDLprotein as previously described.

The foregoing specification, including the specific embodiments, isintended to be illustrative of the present invention and is not to betaken as limiting. Numerous other variations and modifications can beeffected without departing from the true spirit and scope of the novelconcepts of the invention.

What is claimed is:
 1. A hybridoma having the ATCC accession number HB8746.
 2. An antibody that (a) immunoreacts with apoprotein B-100, and(b) has an antibody combining site secreted by the hybridoma having theATCC accession number HB
 8746. 3. The antibody of claim 2 that is anintact antibody.
 4. A cell culture comprising:(a) the hybridoma havingATCC accession number HB 8746; (b) antibody molecules secreted by saidhybridoma that immunoreact with apoprotein B-100; and (c) a culturemedium for said hybridoma.
 5. The culture of claim 19 wherein saidantibody is in substantially pure form.
 6. A sterile affinity sorbantcomprised of biologically active antibody molecules that immunoreactwith apoprotein B-100 and are secreted by hybridoma HB 8746, whereinsaid antibody molecules are affixed to a solid matrix to form a solidsupport.